Production of polyketides

专利摘要:
Recombinant Myxococcus host cells can be used to prepare polyketides, including epothilones and epothilone analogs that can be purified and crystallized from fermentation broth.
公开号:KR20030032942A
申请号:KR1020027014529
申请日:2001-04-26
公开日:2003-04-26
发明作者:로버트 엘. 아슬라니안；개리 애쉴리；스코트 프릭먼；브라이언 쥴리안；레오나드 캇쯔；챠이탄 코슬라；재니스 라우；피터 제이. 리카리；라이카 레젠틴；다니엘 산티；리 탕
申请人:코산 바이오사이언시즈, 인코포레이티드；
IPC主号:

专利说明:

Production method of polyketide {PRODUCTION OF POLYKETIDES}
[2] [2] Polyketides consist of a diverse group of compounds, synthesized at least in part from two carbon unit building block compounds, through a series of Claisen-type condensation reactions and subsequent modifications. Polyketides include antibiotics such as tetracycline and erythromycin, anticancer agents such as epothilones and daunomycin, and immunosuppressive agents such as FK506, FK520 and rapamycin. Polyketides occur naturally in many types of life, including fungi and mycelium. Polyketides are synthesized in vivo by polyketide synthase enzymes, commonly called PKS enzymes. There are two important PKSs known in PKS that differ in their structure and synthesis of polyketides. These two types are commonly referred to as type I or modular PKS enzymes and type II or repeat (aromatic) PKS enzymes.
[3] [3] The present invention provides methods for the production of modular and repeating PKS enzymes, recombinant expression vectors and host cells, and polyketides produced by these enzymes. Modular PKS enzymes are generally multi-protein complexes, where each protein contains a plurality of active sites, each of which is used only once during carbon chain assembly and modification. Repetitive PKS enzymes, on the other hand, are also generally polyprotein complexes, where each protein contains only one or at most two active sites, each of which is used several times during carbon chain assembly and modification. As detailed below, a number of genes were cloned for both modular and directional PKS enzymes.
[4] [4] Modular PKS genes consist of a loading module, several extender modules and coding sequences organized to encode the release domain. As described in more detail below, each of these domains and modules corresponds to a polypeptide having one or more specific functions. In general, the loading module combines with the first building block used to synthesize the polyketide and serves to deliver it to the first extender module. The building blocks used to form the composite polyketide are generally acylthioesters, most often acetyl, propionyl, malonyl, methylmalonyl, hydroxymalonyl, methoxymalonyl and ethylmalonyl CoA. Other building blocks include amino acids and amino acid-like acylthioesters. PKS catalyzes the biosynthesis of polyketides through repeated decarboxylation Claisen condensation reactions between acylthioester building blocks. Each module serves to combine building blocks, to perform one or more functions on that building block, and to deliver the resulting compound to the next module. The next module, in turn, attaches the next building block and delivers the growing compound to the next module until the synthesis is complete. At this point, the polyketide is separated from the PKS by the activity of the release domain, often enzymatic thioesterase (TE).
[5] [5] Polyketides known as 6-deoxyerythronolide B (6-dEB) are synthesized by the prototypical module PKS enzyme. Genes known as eryAI, eryAII and eryAIII encoding multi-subunit proteins known as deoxyerythronolide B synthase or DEBS that synthesize 6-dEB are described in US Pat. Nos. 5,672,491, 5,712,146 and 5,824,513 Which are incorporated herein by reference.
[6] [6] The loading module of DEBS PKS consists of acyltransferase (AT) and acyl carrier protein (ACP). The AT of the DEBS loading module recognizes propionyl CoA (other loading modules AT may recognize other acyl-CoAs such as acetyl, malonyl, methylmalonyl, or butyryl CoA) and as a thioester Pass it to the ACP. At the same time, the AT of each of the six extender modules of DEBS recognizes methylmalonyl CoA (other extender modules ATs are malonyl or alpha-substituted malonyl CoA, ie malonyl, ethylmalonyl and 2-hydroxymalo). Other CoAs, such as Neil CoA), may be passed to the ACP of the module to form thioesters. Once DEBS is primed with propionyl- and methylmalonyl-ACP, the acyl groups of the loading module move to form thioesters in the KS of the first extender module (ester-conversion); In this step, module 1 has acyl-KS adjacent to methylmalonyl ACP. Acyl groups derived from DEBS loading modules are propelled by ancillary decarboxylation, which now covalently binds to the alpha-carbons of the extender groups to form carbon-carbon bonds, which are two carbons longer than the loading unit. Generate a new acyl-ACP with (back or extended) backbone. Growing polyketide chains are transferred from the ACP to the KS of the next module of DEBS, and this process continues in this way.
[7] [7] Polyketide chains grown by the two carbons of each module of DEBS are transferred in series-like processes, from module to module, as covalently bound thioesters. The carbon chain produced by this process alone produces polyketones by filtering out the carbon atoms one by one and having ketones, where the name polyketide is derived, but usually, before the chain is transferred to the next module Additional enzymatic activity is modified by the beta keto group of the polyketide chain to which the 2-carbon unit has been added in advance. Thus, in addition to the minimal modules containing ACP, KS and AT necessary to form carbon-carbon bonds, the modules may contain ketoreductases (KR) that reduce the beta-keto groups to alcohols. The module may also contain KR and a dehydratase (DH) which dehydrates the alcohol in a double bond. The module may also contain KR, DH, and enoylreductase (ER), which converts double bonds into saturated single bonds using beta carbons as methylene functional groups. DEBS molecules include them only with KR domains, only with inactive KR domains, and with all three of the KR, DH and ER domains.
[8] [8] Once the polyketide chain crosses the last module of PKS, it encounters thioesterases found in the release domain, usually at the carboxyl terminus of most PKS enzymes. Here, polyketides are separated from the enzyme, and most, if not all, polyketides are cyclized. Polyketides can be further modified by tailoring or modifying enzymes; These enzymes add carbohydrate groups or methyl groups or add other modifications, ie oxidation or reduction on the polyketide core molecule and / or substituents thereof. For example, 6-dEB is hydroxylated and glycosylated (glycoside screen) and, a glycosyl substituents are methylated saccharide in Indianapolis Fora Erie bitter ah (Saccharopolyspora erythraea) cells, known to be produced by natural antibiotic in Erie Produces thromycin A.
[9] [9] The above description generally applies to modular PKS enzymes, in particular DEBS, and there are a large number of modifications in nature. For example, many PKS enzymes contain loading molecules containing "inactive" KS domains that act as decarboxylase, unlike loading modules of DEBS. This inactive KS is called KS ^Q in most cases, where the superscript is a single letter abbreviation of the amino acid (glutamine) present in place of the active site cysteine required for ketocinase activity. The epothilone PKS loading module contains a KS ^Y domain in which tyrosine is present instead of the active site cysteine. In addition, the synthesis of other polyketides is initiated by different starter units than those bound by DEBS or epothilone loading modules. Enzymes that bind to these starter units include, for example, AMP ligase such as those used for biosynthesis of FK520, FK506 and rapamycin, and non-rebisiomal peptide synthase (NRPS) such as those used for biosynthesis of reinamycin. ), Or soluble CoA ligase.
[10] [10] Another important modification of the PKS enzyme is related to the Bilton block type, which is incorporated as an extender unit. In the case of starter units, one or more NRPS modules are used as extender modules to incorporate amino-like acylthioester building blocks. Epothilone PKS contains, for example, NRPS modules. Another such variant is found in the FK506, Fk520 and rapamycin PKS enzymes, which contain NRPS which acts as the release domain of PKS while incorporating pipecolate residues. Another variant relates to additional activity in the extender module. For example, one module of epothilone PKS contains a methyltransferase (MT) domain that encodes a methyl group to a polyketide.
[11] [11] Recombinant methods for engineering modular and repetitive PKS genes are described in US Pat. 5,672,491; 5,712,146; 5,830,750; And 5,843,718; And PCT Patent Publication Nos. 98/49315 and 97/02358, which are incorporated herein by reference. This and other patent documents disclose recombinant expression for heterologous production of polyketides, as well as recombinant PKS genes assembled by joining two or more different PKS constituents or gene cluster portions to produce novel polyketides. A vector is also disclosed. Streptomyces producing microorganism, and the microorganism is naturally known or novel poly Kane tied in such as not to produce poly Kane Tide E. coli, and yeasts such as (Streptomyces) to produce, naturally it poly Kane Tide as far The methods described above have been used for this purpose (US Pat. No. 6,033,883, incorporated herein by reference). In the latter host, polyketide production is dependent on heterologous expression of phosphopantetheinyl transferase that activates the ACP domain of PKS (see PCT Publication No. 97/13845. See herein). Inserted).
[12] [12] While these methods are valuable and very useful in their own right, some polyketides are only expressed at extremely low concentrations or are toxic to the heterologous host cells used. For example, heterologous expression of the epothilone PKS gene produced anticancer agents epothilones A, B, C and D in Streptomyces (Tang et al., Jan. 28, 2000, Cloning and heterologous expression of the epothilone gene). cluster, Science, 287: 640-642, and PCT Publication No. 00/031247, each incorporated herein by reference). Epothilones A and B were produced at concentrations below 50-100 μg / L and have been shown to adversely affect producer cells.
[13] [13] Epothilones A and B were initially identified as having antifungal activity derived from Sorangium cellulosum , Myxobacterium (Gerth et al., 1996, J. Antibiotics 49: 560-563 and Germany). See DE 41 38 042, incorporated herein by reference, and later found to have activity in tubulin polymerization assays (Bollag et al., 1995, Cancer Res. 55: 2325-2333, incorporated herein by reference) ). Since then, epothilones A and B and certain naturally occurring derivatives and synthetic derivatives have been intensively studied as potential antitumor agents for the treatment of cancer. The chemical structure of the epothilones produced by the Sorangium cellulosum strain So ce 90 is described in Hofle et al., 1996, Epothiolone A and B-novel 16-membered macrolides with cytotoxic activity: isolation, crystal structure, and conformation in solution , Angew. Chem. Int. Ed. Engl. 35 (13/14): 1567-1569 (incorporated herein). Epothilone A (R = H) and B (R = CH ₃ ) have the following structure and have been shown to have broad cytotoxic activity against eukaryotic cells and prominent activity and selectivity against breast tumor and colon tumor cell lines.
[14]
[15] [14] Solanzium cellulose , although the desoxy counterparts of epothilones A and B, also known as epothilones C (R = H) and D (R = CH ₃ ), have been chemically synthesized by the de novo method. (Sorangium cellulosum ) is also present as a small amount of product when epothilones C and D are less cytotoxic than epothilones A and B; The structure is as follows.
[16]
[17] [15] Other naturally occurring epothilones have also been described. These include epothilones E and F, wherein the methyl side chains of the thiazole moieties of epothilones A and B are hydroxylated to produce many other epothilone compounds as well as epothilones E and F, respectively (PCT Publication No. 99 / 65913, incorporated in the text)
[18] [16] Epothilones have potential uses as anticancer agents, and because of the low concentrations of epothilones produced by the natural Soce 90 strain, several teams have attempted to synthesize epothilones. As noted above, this effort has been successful (Balog et al., 1996, Total synthesis of (−)-epothilone A, Angezv. Chem. Int. Ed. Engl. 35 (23/24): 2801-2803; Su Et al., 1997, Total synthesis of (-)-epothilone B: an extension of the Suzuki coupling method and insights into structure-activity relationships of the epothilones, Angew.Chem.Int.Ed . Engl. 36 (7): 757- 759; Meng et al., 1997, Total syntheses of epothilones A and B, JACS 119 (42): 10073-10092; and Balog et al., 1998, A novel aldol condensation with 2-methyl-4-pentenal and its application to an improved total synthesis of epothilone B, Angew.Chem.Int.Ed.Eng . 37 (19): 2675-2678, all incorporated herein by reference). Despite the success of these efforts, chemical synthesis of epothilones is tedious, time consuming and expensive. Indeed, this method is useless for large-scale pharmaceutical development of epothilones as anticancer agents.
[19] [17] A number of epothilone derivatives, as well as epothilones A-D, have been studied in vitro and in vivo (Su et al., 1997, Structure-activity relationships of the epothilones and the first in vivo comparison with paclitaxel, Angew. Int.Ed. Engl. 36 (19): 2093-2096; and Chou et al., Aug. 1998, Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B, Proc. Natl. Acad. Sci. USA 95 : 9642-9647, incorporated herein by reference). Additional epothilone derivatives and methods for the synthesis of epothilones and epothilone derivatives are described in PCT Publication Nos. 00/23452, 00/00485, 99/67253, 99/67252, 99/65913, 99/54330, 99/54319, 99/54318 , 99/43653, 99/43320, 99/42602, 99/40047, 99/27890, 99/07692, 99/02514, 99/01124, 98/25929, 98/22461, 98/08849, and 97/19086; U.S. Patent No. 5,969,145; And in German Patent Publication DE 4138 042, which is incorporated herein by reference.
[20] [18] There remains a need for economic means for producing naturally occurring epothilones, derivatives or precursors thereof, as well as novel epothilone derivatives with improved properties. There is a need for host cells that produce epothilones or epothilone derivatives and produce more desired polyketide products more easily to handle and ferment than this natural producer Sorangium cellulosum. The present invention meets the above needs by providing high levels of polyketide and providing host cells useful for producing not only epothilones, but also the novel epothilone derivatives and other polyketides described herein.
[21] Summary of the Invention
[22] [19] In one embodiment, the invention containing a recombinant expression vector encoding a heterologous PKS gene in cis tobak Te Linea producing poly Kane Tide synthesized by encoding PKS enzymes by the genes on these vectors ( Cystobacterineae ) provides a recombinant host cell of the subfamily. In a preferred embodiment, the host cell is derived from genus Rhodococcus mikso (Myxococcus) in or stigmasterol telra (Stigmatella). In a particularly preferred embodiment, the host cell comprises M. stipitatus, M. fulvus, M. xanthus, M. xanthus, M. virescens, It is preferred to be selected from S. ectecta, and S. aurantiaca .
[23] [20] In another embodiment, the present invention provides a recombinant DNA vector capable of chromosomal insertion or extrachromosomal replication in a host cell of the present invention. Vectors of the invention comprise at least a portion of a PKS coding sequence and can direct expression of a functional PKS enzyme in a host cell of the invention. In a related embodiment, the present invention provides host cells and vectors comprising genes or gene products required to produce substrates of polyketide biosynthesis that are not produced in the host cells of the invention or are produced only in small amounts. In one embodiment, the gene and gene product catalyze the synthesis of ethylmalonyl CoA. In another embodiment, the genes and gene products catalyze the synthesis of butynyl CoA.
[24] [21] In another embodiment, the present invention provides a method for producing polyketide in a host cell of Cystobacterineae subtype, which is not naturally produced in the host cell. The method includes culturing the host cell transformed with the recombinant DNA vector of the present invention under the condition that the PKS gene coated on the vector is expressed, and then producing the polyketide. In a related embodiment, the present invention provides a method of fermenting a host cell of the present invention that enables high yield production of polyketides.
[25] [22] In a preferred embodiment, the recombinant host cell of the present invention produces an epothilone or an epothilone derivative. Accordingly, the present invention provides recombinant host cells that produce the desired epothilones or epothilone derivatives. In a preferred embodiment, the host cell produces one or more epothilones in an amount of at least 10 mg / L. In one embodiment, the present invention provides host cells that produce one or more epothilones in greater amounts than wool produced in organisms that naturally produce epothilones. In another embodiment, the present invention provides host cells that produce mixtures of epothilones that are less complex than the mixtures produced by naturally occurring host cells producing epothilones. Recombinant host cells of the invention also include host cells that produce only one desired epothilone or epothilone derivative as the main product.
[26] [23] In a related preferred embodiment, the present invention provides recombinant DNA expression vectors encoding some or all of epothilone PKS. Accordingly, the present invention provides recombinant DNA expression vectors encoding the proteins required to produce epothilones A, B, C and D in the host cells of the present invention. The present invention also provides recombinant DNA expression vectors encoding these proteins. The present invention also provides a recombinant DNA compound encoding a hybrid protein comprising all or part of a protein involved in epothilone biosynthesis and all or part of a protein associated with biosynthesis of another polyketide or ribosomal-derived peptide.
[27] [24] In another embodiment, the present invention provides novel epothilone derivative compounds in substantially pure form useful in the fields of agriculture, veterinary medicine and medicine. These compounds include 16-desmethyl of epothilones A, B, C and D; 14-methyl; 13-oxo; 13-oxo-11,12-dehydro; 12-ethyl; 13-hydroxy-10,11-dehydro; 11-oxo; 11-hydroxy; 10-methyl; 10,11-dehydro; 9-oxo; 9-hydroxy; 8-desmethyl; 6-desmethyl; And 2-methyl analogues, and various analogs in which the methylthiazole portion of the naturally occurring epothilone is replaced with another portion. In one embodiment, these compounds are useful as fungicides. In another embodiment, these compounds are useful for chemotherapy of cancer as anticancer agents. In a preferred embodiment, the compound is an epothilone derivative that has at least as strong activity as epothilone B or D on tumor cells. In another embodiment, these compounds are useful as immunosuppressive agents. In another embodiment, these compounds are useful for the preparation of another compound. In a preferred embodiment, these compounds may be formulated as a mixture or solution for administration to humans or animals.
[28] [25] In another embodiment, the present invention provides a method for purifying epothilone. In a preferred embodiment, the epothilone is preferably purified from fermentation broth.
[29] [26] In another embodiment, the present invention provides the epothilone compound in a highly purified form. In a preferred embodiment, the epothilones have a purity of greater than 95%. In a more preferred embodiment, the epothilones are more than 99% pure. In a particularly preferred embodiment, the present invention provides the epothilone in crystalline form. In a particularly preferred embodiment, the present invention provides crystalline epothilone D.
[30] [27] In another embodiment, the present invention provides a treatment for cancer comprising administering a therapeutically effective amount of the novel epothilone compound of the present invention. The compounds and compositions of the present invention are also effective in treating other proliferative diseases or conditions, including but not limited to psoriasis and inflammation.
[31] These and other embodiments of the invention will be described in more detail in the following description, examples, and claims.
[1] [1] The present invention provides recombinant methods and materials for producing polyketides in recombinant host cells; Recombinant host cells producing polyketides; Novel polyketides structurally associated with epothilones; Purification method of epothilone; And crystalline form of epothilone D. In a preferred embodiment, the recombinant host cell of the invention is transformed with a recombinant DNA expression vector of the invention encoding a modular or iterative polyketide synthase (PKS) gene, system tobak to Te in Linea (Cystobacterineae) suborder, preferably is derived from the genus Rhodococcus mikso (Myxococcus) and stigmasterol in telra (Stigmatella). The recombinant host cells of the present invention produce and are capable of producing known and novel polyketides including, but not limited to, epothilones and epothilone derivatives. The present invention relates to the fields of agriculture, chemistry, medicinal chemistry, medicine, molecular biology and pharmacology.
[377] [29] Figure 1 shows several of the N-acetyl cystiamine thioester derivatives that can be fed to the epothilone PKS of the invention where the NRPS-like Module 1 or Module 2 KS domains are inactivated to produce novel epothilone derivatives. Show precursor compounds. The general synthesis process for making such compounds is also shown.
[378] 2 shows restriction map and functional map of plasmids pKOS35-82.1 and pKOS35-82.2.
[379] [31] Figure 3 shows the restriction site and function map of the plasmids pKOS35-154 and pKOS90-22.
[380] [32] Figure 4 illustrates a schematic diagram of the protocol and formula gene for introducing the epothilone PKS into the chromosome of Lactococcus mikso glass tooth (Myxococcus xantus) host cells as described in Example 2.
[381] [33] Figure 5 shows a map of pBeloBACII as described in Example 2.
[382] [34] Figure 6 is shows the baseline performance of 5g / L shi tone (pancreatic casein digest) and 2g / L magnesium mikso Lactococcus glass in a simple production medium consisting only sulfate tooth (Myxococcus xantus) K111-40-1 . Sign Description: Growth (●), Production (■), and Ammonia Generation (▲) in basal CTS medium in a batch process; Culture conditions are as described in the Materials and Methods column of Example 3.
[383] [35] Figure 7 shows the effect of XAD-16 resin on the fermentation performance of Lactococcus mikso glass tooth (Myxococcus xantus) strain K111-40-1 in CTS production medium. Explanation: Growth (●) and Production (■) Profiles with 20 g / L of XAD-16 resin added to the CTS production medium in the batch process.
[384] [36] Figure 8 shows the effect of carcitone on growth and production yield. Code Description: The effect of carcitone concentration on growth (●), production (■), and specific productivity (▲).
[385] 9 shows the effect of trace elements and high concentrations of methyl oleate on growth and production yields. Code Description: Effects of methyl oleate (●) and trace elements (■) on production.
[386] And [38] Figure 10A shows the growth and production of Lactococcus mikso glass tooth (Myxococcus xantus) strain if the methyl oleate and trace elements in a batch fermentation process, the optimum concentration exists. The log phase occurred within the first two days of inoculation. Production of epothilone D started with the onset of normal phase and ceased when cell lysis occurred due to depletion of methyl oleate on day 5. 10B is corresponding to the consumption in the case of the methyl oleate and trace elements in a batch fermentation process, present in optimal concentration mikso Lactococcus glass tooth (Myxococcus xantus) of ammonia during the growth and production of the strain proceeds generated and methyl oleate Show time lapse. Code Description: A) Growth (●) and Production (■) Profiles when the optimum concentration of methyl oleate (7 mL / L) and trace elements (4 mL / L) was added to the CTS production medium in the batch process. B) Time course corresponding to consumption amount of methyl oleate (●) and ammonia generation amount (■).
[387] Shows the influence of the batch process (intermittent fed-batch process) - [39] Figure 11A is mikso Lactococcus glass tooth (Myxococcus xantus) intermittent injection on the growth and production of the strain. Code Description: Growth (●) and Production (■) Profiles in Intermittent Infusion-Batch Processes in Shake-Flasks. The rates of infusion of citaton and methyl oleate were 2 g / L / 1 day and 3 mL / L / 1 day, respectively. 11B shows the generation of ammonia and the constant consumption rate of methyl oleate during fermentation. Explanation of symbols: Time course corresponding to the total amount of methyl oleate added to the medium (●), the total consumption of methyl oleate (■), and the amount of ammonia generated (▲).
[388] [40] Figure 12 shows the production profile for the intermittent injection-batch process in 5-L bioreactors. The infusion rates of carcitone and methyl oleate were 2 g / L / 1 day and 3 mL / L / 1 day, respectively.
[389] [41] Figure 13A shows the effect of continuous infusion on growth and production. Explanation: Growth (●) and Production (■) Profiles in a Continuous Injection-Batch Process in 5-L Bioreactors. The infusion rates of carcitone and methyl oleate were 2 g / L / 1 day and 3 mL / L / 1 day, respectively. 13B shows the time course for the addition and consumption of methyl oleate as well as the amount of ammonia generated during the continuous infusion-batch process. Explanation of symbols: Time course corresponding to the total amount of methyl oleate added to the medium (●), the total consumption of methyl oleate (■), and the amount of ammonia generated (▲). Culture conditions are as described in the Materials and Methods column.
[32] The scope of the present invention and the definitions of terms used herein are set forth below. Unless otherwise defined in a particular case, these definitions apply to the terms as used individually or as part of a larger scope throughout this specification.
[33] [43] All stereoisomers of the compounds of the present invention as well as the pure compounds of the present invention and mixtures thereof are included in the scope of the present invention. Individual enantiomers, diastereomers, geometric isomers, and combinations and mixtures thereof are all within the scope of the present invention. In addition, some of the crystalline forms of these compounds may exist as polymorphs, and are included in the present invention as they are. In addition, some of these compounds may form solvates with water (ie, hydrates), or may form solvates with common organic solvents, and such solvates are also encompassed within the scope of the present invention.
[34] [44] Protected forms of the compound of the present invention are also included in the scope of the present invention. Various protecting groups are described, for example, in T.H. Greene and P.G.M. Illustrated in Wuts, Protective Groups in organic Synthesis, 3rd edition, John Wiley & Sons, New Yokr (1999), which is incorporated herein by reference. For example, the hydroxy protected form of the compounds of the invention is that at least one of the hydroxy groups is protected by a hydroxy protecting group. Examples of hydroxy protecting groups include tetrahydropyranyl; benzyl; Methylthiomethyl; Ethylthiomethyl; Pivaloyl; Phenylsulfonyl; Triphenylmethyl; Tri-substituted such as trimethyl silyl, triethylsilyl, tributylsilyl, tri-isopropylsilyl, t-butyldimethylsilyl, tri-t-butylsilyl, methyldiphenylsilyl, ethyldiphenylsilyl, t-butyldiphenylsilyl, etc. Silyl; And aroyl such as acyl and acetyl, pivaloylbenzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryl, and the like. Keto groups of the compounds of the invention can also be protected in a similar manner.
[35] [45] The scope of the present invention also includes precursors of the compounds of the present invention. In general, such precursors are functional derivatives of the desired compound that can be readily converted to the desired compound in vivo. Thus, the term "administering" in the methods of treatment of the present invention refers to a compound that is explicitly described herein or a compound that is not explicitly described, or that is specified in vivo after administration to a patient for the treatment of various diseases. It also includes treatment with the compound to be converted. Conventional methods for the selection and preparation of suitable precursor derivatives are described, for example, in "Design of Prodrugs", H. Bundgaard, Elsevier, 1985.
[36] [46] As used herein, the term "aliphatic" refers to saturated and unsaturated straight, branched, cyclic, or polycyclic hydrocarbons which may be optionally substituted in one or more positions. Examples of cycloaliphatic groups include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl moieties. The term "alkyl" refers to a straight or branched chain saturated hydrocarbon substituent. "Alkenyl" refers to a straight or branched chain hydrocarbon substituent with at least one carbon-carbon double bond. "Alkynyl" refers to a straight or branched chain hydrocarbon substituent having at least one carbon-carbon triple bond.
[37] [47] The term "aryl" refers to a monocyclic or polycyclic group having at least one aromatic ring structure, optionally comprising one or more heteroatoms and preferably 3 to 14 carbon atoms. Aryl substituents may be optionally substituted at one or more positions. Examples of aryl groups are: furanyl, imidazolyl, indanyl, indenyl, indolyl, isoxazolyl, isoquinolinyl, naphthyl, oxazolyl, oxdiazolyl, phenyl, pyrazinyl, pyridyl, pyri Midinyl, pyrrolyl, pyrazolyl, quinolyl, quinoxalyl, tetrahydronaphthyl, tetrazoyl, thiazoyl, thienyl, thiophenyl, and the like.
[38] [48] The alicyclic (ie, alkyl, alkenyl, etc.) and aryl moieties may include one or more substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents. Optionally substituted by The definition of any substituent or variable at any location in the molecule is irrelevant to its definition elsewhere in the molecule. Substituents and substitution patterns on the compounds of the present invention may be selected by those skilled in the art to be readily synthesized by known techniques as well as the methods presented herein and to provide chemically stable compounds. Examples of suitable substituents include: alkyl, alkenyl, alkynyl, aryl, halo; Trifluoromethyl; Trifluoromethoxy; Hydroxy; Alkoxy; Cycloalkoxy; Heterocyclooxy; Oxo; Alkanoyl (-C (= 0) -alkyl, also referred to as "acyl"), aryloxy; Alkanoyloxy; Amino; Alkylamino; Arylamino; Aralkylamino; Cycloalkylamino; Heterocycloamino; Two amino substituents are di-substituted amines selected from alkyl, aryl or aralkyl; Alkanoylamino; Aroylamino; Aralkanoylamino; Substituted alkanoylamino; Substituted arylamino; Substituted aralkanoylamino; Thiols; Alkylthio; Arylthio; Aralkylthio; Cycloalkylthio; Heterocyclothio; Alkylthioo; Arylthiono; Aralkylthionos; Alkylsulfonyl; Arylsulfonyl; Aralkylsulfonyl; Sulfonamido (such as SO ₂ NH ₂ ); Substituted sulfonamido; Nitro; Cyano; Carboxy; Carbamyl (eg, CONH ₂ ); Substituted carbamyl (eg, —C (═O) NRR ′ where R and R ′ are each independently hydrogen, alkyl, aryl, aralkyl, etc.); Heterocyclo, such as alkoxycarbonyl, aryl, substituted aryl, guanidino and indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl, and the like. .
[39] [49] "Alkylaryl" or "arylalkyl" refers to an aryl group having an aliphatic substituent bonded to the compound via an aliphatic group. An example of an alkylaryl-substituted arylalkyl group is benzyl, ie phenyl having a methyl group bonded to the compound via a methyl group (ie, —CH ₂ PH where Ph is phenyl).
[40] [50] The term "acyl" refers to -C (= 0) R, where R is an aliphatic group, preferably a C ₁ -C ₆ moiety.
[41] [51] The term "alkoxy" refers to -OR, where O is oxygen and R is an aliphatic group.
[42] [52] "Aminoalkyl" refers to -RNH ₂ , wherein R is an aliphatic moiety.
[43] [53] "Halogen", "halo", or "halide" refers to fluorine, chlorine, bromine and iodine.
[44] [54] "Haloalkyl" is -RX, wherein R is an aliphatic moiety and X is one or more halogen.
[45] [55] "hydroxyalkyl" is -ROH, wherein R is an aliphatic moiety.
[46] [56] "Oxo" refers to carbonyl oxygen (= 0).
[47] [57] In addition to the substituents specified in the foregoing groups, the compounds of the present invention may also include other substituents as appropriate. For example, the lactam or lactam skeleton or skeletal substituent may be additionally substituted with one or more substituents, such as C ₁ -C ₅ aliphatic, C ₁ -C ₅ alkoxy, aryl or functional groups (eg, replacing one of hydrogen or By inducing non-hydrogen groups). Examples of suitable functional groups are: acetal, alcohol, aldehyde, amide, amine, boronate, carbamate, carboalkoxy, carbonate, carbodiimide, carboxylic acid, cyanohydrin, disulfide, enamine, ester, ether, halogen, Hydrazide, hydrazone, imide, imido, imine, isocyanate, ketal, ketone, nitro, oxime, phosphine, phosphonate, phosphoric acid, quaternary ammonium, sulfphenyl, sulfide, sulfone, sulfonic acid, thiol, etc. But may not be limited thereto.
[48] In the present specification, the use of the term “isolated” with respect to the compound of the present invention means that it is changed by human interference from its original natural state. For example, if a compound occurs naturally, it may be altered or removed from its original environment, or both. That is, a compound present in a living organism is not "isolated", but according to an application herein, the same compound that is separated from its natural state of coexistence is "isolated". The term "isolated" also means compounds in the preparation that are substantially free of impurities or unwanted substances. In the case of a compound found in nature, it means that in its original state there is actually no substance associated with the compound or composition.
[49] [59] The term "purified" for a compound means that the compound is present in the formulation as a main component, i.e., about 505, about 60%, about 705, about 80%, about 90 relative to the total weight of the formulation. %, By weight of at least about 95%.
[50] [60] As used herein, the term "subject" refers to an animal, preferably a mammal, to be treated, observed or tested, most preferably a human being treated and / or observed. Point.
[51] [61] A "therapeutically effective amount" means an amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue system, animal or human being explored by a researcher, veterinarian, physician or other clinician. And includes alleviating the symptoms of the disease or disorder being treated.
[52] [0053] The term "composition" includes not only a specific amount of any product resulting directly or indirectly from a combination of specific components, but also a product containing a specific amount of specific components.
[53] [63] "Pharmaceutically acceptable salt" refers to one or more salts of the compound of the present invention. Suitable pharmaceutically acceptable salts of the compounds of the invention include, for example, solutions of pharmaceutically acceptable acids such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carboxylic acid or phosphoric acid with a solution of the compound. Acid addition salts that can be formed by mixing are included. In addition, when the compound of the present invention has an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts (such as sodium or potassium salts); Alkaline earth metal salts (such as calcium or magnesium salts); And salts formed with suitable organic ligands (e.g., counter anions such as ammonium, quaternary ammonium and halides, hydroxides, carboxylates, sulfates, phosphonates, nitrates, alkylsulfonates and aryl sulfonates) Amine cation). Examples of pharmaceutically acceptable salts include: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitatrate, borate, bromide, butyrate, calcium edetate, Camphorates, camphorsulfonates, campylates, carbonates, chlorides, citrates, clavulanates, cyclopentanepropionates, digluconates, dihydrochlorides, dodecylsulfates, edetates, edisylates, estolates, Ecylate, ethanesulfonate, formate, fumarate, glutamate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolasanilate, hemisulfate, heptanoate, hexanoate, hexylresorcis Nates, hydrabamine, hydrobromide, hydrochloride, Droiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, maleate, maleate, malonate , Mandelate, mesylate, methanesulfonate, mesyl sulfate, mucate, 2-naphthalenesulfonate, lead sillate, nicotinate, nitrate, N-methylslucamine ammonium salt, oleate, oxalate, pamoate ( Embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalalturonate, propionate, salicylate, stearate Latex, sulfate, subacetate, succinate, tannate, tartrate, theocatelate, tosylate, triethiodide, undecanoate, valerate, etc. It includes and is not limited to this.
[54] [64] "Pharmaceutically acceptable carrier" refers to the medium used to prepare the intended dosage form of the compound of the invention. Pharmaceutically acceptable carriers include solvents, diluents or other liquid vehicles; Dispersion or suspension aids; Surfactants; Isotonic agents; Thickening or emulsifying agents, preservatives; Solid binders; Lubricants and the like. Remington's Pharmaceutical Sciences, 15th edition, EW Martin (Mack Publishing Co., Easton, Pa, 1975) and Handbook of Pharmaceutical Excipients, 3rd edition, AH Kibbe, Part (Amer. Pharmaceutical Assoc. 2000), are used to prepare pharmaceutical compositions. Various carriers and known techniques for their preparation have been described, both of which are incorporated herein by reference.
[55] [0065] "Pharmaceutically acceptable ester" refers to an ester that is hydrated in vivo and converted into a compound of the present invention or a salt thereof. Examples of suitable ester groups include those derived from pharmaceutically acceptable aliphatic carboxylic acids such as formate, acetate, propionate, butyrate, acrylate and ethyl succinate.
[56] [66] The present invention provides a recombinant method and material for producing polyketide in recombinant host cells; Recombinant host cells producing polyketides; Novel polyketides structurally related to epothilones; Purification method of epothilone; And crystalline form of epothilone D.
[57] [67] In one embodiment, the present invention is cis tobak Te Linea producing poly Kane Tide synthesized by a PKS enzyme encoded on said vector contains an expression vector encoding the (heterologous) PKS gene heterologous ( Cystobacterineae ) provides a recombinant host cell of the subfamily. As used herein, recombination is a term used for cells, compounds, or compositions produced by direct manipulation of genes or portions thereof by human intervention, typically directly. The system tobak Te Linea (Cystobacterineae) Amok is one of two that belong to Mick Socorro Karl Les (Myxococcales) neck (the other one as the turmoil centipede ah (Sorangineae), This turmoil that produce epothilones erase cellulite Rossum (Sorangium cellulosum )). System is a tobak Te Linea (Cystobacterineae) suborder includes the on and (Myxococcaceae) in mikso Coca Seah and cis tobak Te Linea (Cystobacterineae). Mikso Coca-year olds (Myxococcaceae) and are not geo Caucus (Angiococcus) in (ie, A. disciformis), Caucus (Corallococcus) in (that is, mikso Caucus (Myxococcus), A coral back to C. macrosporus, C. corralloides, And C. exiguus ). The cis tobak Te Linea (Cystobacterineae) and has Sisto bakteo (Cystobacter), A (i.e., C. fuscus, C. ferrugineus, C. minor, C. velatus, and C. violaceus), mellitic carbon Clear (Melittangium) in include (i. e., M. and M. boletus lichenicola), stigmasterol telra (Stigmatella) in (i.e., S. erecta and S. aurantiaca), and are square wipe (Archangium) in (i. e., A. gephyra). Particularly preferred host cells in the present invention are those which produce 10 to 20 mg / L or more, more preferably 100 to 200 mg / L or more, most preferably 1 to 2 g / L or more of polyketide.
[58] [68] In a preferred embodiment, the host cells of this invention derived from the genus Rhodococcus mikso (Myxococcus) in or stigmasterol telra (Stigmatella). In a particularly preferred embodiment, the host cell is selected from M. stipitatus, M. fulvus, M. xanthus, M. virescens, S. erecta and S. aurantiaca . Particularly preferred myxococcal host cells of the invention have a polyketide of at least 10-20 mg / L, more preferably 100-. 200 mg / L or more, most preferably 1 to 2 g / L or more. Especially preferred are M. xanthus host cells producing at this concentration. M. xanthus host cells usable for the purposes of the present invention include DZ1 cell line (Campos et al., 1978, J. Mol. Biol. 119: 167-178, incorporated herein by reference), TA-producing cell line ATCC 31046, DK1219 Cell lines (Hodgkin and Kaiser, 1979, Mol. Gen. Genet. 171: 177-191, incorporated herein by reference) and DK1622 cell lines (Kaiser, 1979, Proc. Natl. Acad. Sci. USA 76: 5952-5956, Incorporated herein by reference), but is not limited thereto.
[59] [69] The host cells of the present invention comprise recombinant DNA expression vectors, and in another embodiment, the present invention provides recombinant DNA vectors capable of chromosomal integration or extrachromosomal replication in these host cells. to provide. Vectors of the invention comprise at least a portion of a PKS coding sequence and can direct the expression of a functional PKS enzyme in a host cell of the invention. As used herein, expression vector refers to any nucleic acid that can be introduced into a host cell. Expression vectors can be stably or temporarily maintained in cells as part of a chromosome or other DNA in a cell or compartment of cells, such as a replication vector in the cytoplasm. Expression vectors also include genes that serve to direct synthetic RNA translated into polypeptides in cells or cell extracts. Thus, this vector contains a promoter that enhances gene expression or is inserted into any site within the chromosome so that gene expression is achieved. In addition, since expression vectors generally contain additional functional elements such as resistance-conferring genes, they act as selectable markers and regulatory genes that enhance promoter activity.
[60] In general, an expression vector will contain one or more marker genes such that host cells containing the vector can be identified and / or selected. Examples of antibiotic resistance genes to be used in the vectors of the present invention include ermE ( improve resistance to erythromycin and lincomycin), tsr ( improve resistance to thiostrepton ), aaaA (spectinomycin and streptomycin) Conferred ), aaaC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to biomycin ) ) Resistance conferring gene, but is not limited thereto. Mikso in Lactococcus glass tooth selectable marker used in the (Myxococcus xanthus) can to kanamycin, tetracycline, chloramphenicol, Zeocin, spectinomycin and streptomycin resistance imparting gene.
[61] Various components of the expression vector can vary greatly depending on the desired use of the vector. In particular, the components of the vector are highly dependent on the host cell in which the vector will be used and the intended function of the vector. For example, certain preferred vectors of the invention are insertion vectors: the vector is inserted into the chromosomal DNA of the host cell. Such a vector may comprise a DNA segment complementary to a site of phage attachment or to a segment of chromosomal DNA of a host cell directed to insertion. In addition, as exemplified herein, a series of such vectors can be used to construct a PKS gene cluster in a host cell, where each vector comprises only a portion of a complete PKS gene cluster. Thus, the recombinant DNA expression vector of the invention may comprise only a portion of the PKS gene or gene cluster. Homologous recombination can also be used to remove, destroy or alter genes, including heterologous PKS genes previously introduced into host cells.
[62] In a preferred embodiment, the present invention provides a host cell containing an expression vector and an expression vector that produces a recombinant Myxococcus , preferably Myxococcus xanthus , ie, a polyketide. do. Although there are unpublished reports of artificial plasmids based on Mx4 phage replication, vectors that are extrachromosomal to replicate in Myxococcus xanthus are currently known. However, there are several phages known to be inserted into the chromosomal DNA of Myxococcus xanthus, including Mx8, Mx9, Mx81, and Mx82. Application of these phage integration and attachment functions to plasmids can produce phage-based expression vectors that are inserted into the chromosomal DNA of Myxococcus xanthus . Among these, phage Mx9 and Mx8 are preferable for the purposes of the present invention. Salmi et al., Feb. Plasmid pPLH343, described in Genetic determinants of immunity and integration of temperature Myxococcus xanthus phage Mx8, J. Bact. 180 (3): 614-621), is a plasmid that replicates in E. coli and encodes the attachment and insertion function. Phage Mx8 gene.
[63] [73] There is a wide range of promoters may be used in the preferred mikso Rhodococcus (Myxococcus) expression vector of the present invention. See, eg, Example 8, below. For example, the promoter of the Sorangium cellulosum epothilone PKS gene (see PCT Publication No. 00/031247, incorporated herein by reference) functions in Myxococcus xanthus host cells. Epothilone PKS gene promoters can be used to drive expression of one or more epothilone PKS genes or other PKS gene production in recombinant host cells. Another preferred promoter which can be used in Myxococcus xanthus for the purpose of expressing the recombinant PKS of the present invention is the promoter of the pilA gene of Myxococcus xanthus . pilA promoter, two mikso Lactococcus glass tooth (Myxococcus xanthus) strain pilA deletion strain and pilS deletion strain and the highly expressed promoter that the gene products from genes controlled by the pilA is Wu and Kaiser, Dec. 1997, Regulation of expression of the pil A gene in Myxococcus xanthus, J. Bact. 179 (24): 7748-7758 (incorporated herein). The invention also recombinant Lactococcus mikso (Myxococcus) host cell comprising a deletion of the pilA and pilS both provided. Another preferred embodiment is the hunger dependent promoter of the sdeK gene.
[64] [74] The present invention provides a recombinant Myxococcus xanthus expression vector and a preferred expression vector for use in preparing a host cell. These vectors, designated plasmids pKOS35-82.1 and pKOS35-82.2 (see FIG. 2), can be inserted into the chromosomal DNA of Myxococcus xanthus as well as to replicate E. coli host cells. The vectors conveniently pilA promoter not only include Mx8 attached and inserting the gene having the restriction enzyme recognition site downstream located. These two vectors differ only in the direction of the pilA promoter on the vector and can be easily modified to include the modified target gene of the present invention and the epothilone PKS or other PKS and the modified enzyme gene. The structure of the vector was described in Example 1.
[65] [75] In another embodiment, the present invention in a host cell of the system tobak Te Linea (Cystobacterineae) suborder naturally is to provide a method for producing poly cake tied that are not produced, the method comprising the recombinant DNA of the invention Culturing the host cell transformed with the vector under conditions such that the PKS gene encoded on the vector is expressed to produce a polyketide. By this method it is possible to produce all of the various types of polyketides produced by modular or repeat PKS enzymes. In addition, novel polyketides derived from hybrid or other recombinant PKS genes can also be prepared using this method. In a preferred embodiment, the PKS gene encodes a hybrid modular PKS.
[66] Many modular PKS genes have been cloned and are readily available in the methods and vectors of the invention. Polyketides produced by PKS enzymes can be further modified by polyketide modifying enzymes, called tailoring enzymes, which hydroxylate, epoxidize, methylate and / or glycosylate polyketides. have. According to the methods of the invention, these genes may be introduced into host cells for the production of the desired modified polyketide. The following table shows references describing exemplary PKS and PKS enzymes that can be used to prepare recombinant PKS, and the corresponding DNA compounds encoding them of the invention. Also provided are various references describing tailoring and modifying polyketides and corresponding genes that can be used to make the recombinant DNA compounds of the invention.
[67] PKS and Polyketide Custom Enzyme Genes
[68] Avermectin
[69] [77] US Pat. No. 5,252,474 to Merck; U.S. Patent 4,703,009; And European Publication 118,367.
[70] [78] MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.
[71] [79] MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.
[72] [80] Ikeda and Omura, 1997, Chem. Res. 97: 2599-2609, Avermectin biosynthesis.
[73] Candicidin (FR008)
[74] [81] Hu et al., 1994, Mol. Microbiol. 14: 163-172.
[75] Epothilone
[76] [82] Novatis PCT Publication No. 99/66028
[77] [83] Kosan's PCT Publication No. 00/031247
[78] Erythromycin
[79] [84] Abbott's PCT Publication No. 93/13663; U.S. Patent No. 6,004,787 ;. And US Patent No. 5,824,513.
[80] [85] Donadio et al., 1991, Science 252: 675-9.
[81] [86] Cortes et al., 8 Nov. 1990, Nature 348: 176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea .
[82] Glycosylation Enzymes
[83] [87] Abbott's PCT Publication No. 97/23630 and US Patent No. 5,998,194
[84] FK-506
[85] [88] Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK-506, Eur. J. biochem. 256: 528-534.
[86] [89] Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK-506, Eur. J. Biochem. 244: 74-80.
[87] Methyltransferase
[88] [90] United States Patent No. of Merck. 5,264,355 and US Patent No. 5,622,866.
[89] [91] Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK-520, J. Bacteriol. 178: 5243-5248.
[90] FK-520
[91] [92] Kosan's PCT Publication No. 00/20601.
[92] [93] Nielsen et al., 1991, Bioc 1 zem. 30: 5789-96.
[93] Lovastatin
[94] [94] United States Patent No. of Merck. 5,744,350.
[95] Nemadectin
[96] [95] MacNeil et al., 1993, supra.
[97] Nidamycin
[98] [96] Abbott's PCT Publication No. 98/51695.
[99] [97] Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis , J. Bacteriol. 179: 7515-7522.
[100] Oleandomycin
[101] [98] Swan et al., 1994, Characterization of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Mol. Gen. Genet. 242: 358-362.
[102] [99] Kosan's PCT Publication No. 00/026349.
[103] [100] Olano et al., 1998, Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases' responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259 (3): 299308.
[104] [101] Hoechst's PCT Publication No. 99/05283.
[105] Picromycin
[106] [102] Kosan's PCT Publication No. 99/61599.
[107] [103] PCT Publication No. of the University of Minnesota 00/00620.
[108] [104] Xue et al., 1998, Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae, Chemistry & Biology 5 (11): 661-667.
[109] [1051 Xue et al., Oct. 1998, A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae : Architecture of metabolic diversity, Proc. Natl. Acad. Sci. USA 95: 12111 12116.
[110] Platenolide
[111] [1061 Lilly's EP Publication No. 791, 656; And US Patent No. 5,945,320.
[112] Rapamycin
[113] 1071 Schwecke et al., Aug. 1995, The biosynthetic gene cluster for the polyketide rapamycin, Proc. Natl. Acad. Sci. USA 92: 7839-7843.
[114] [108] Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus : analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.
[115] Rifamycin
[116] [109] Novatis' PCT Publication No. 98/07868.
[117] [110] August et al., 13 Feb. 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry & Biology, 5 (2): 69-79.
[118] Sorangium PKS
[119] [111] US Pat. 6,090,601.
[120] Sorafen
[121] [112] US Patent No. of Novartis 5,716,849.
[122] [113] Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangium cellulosuni (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polysynide Synthase Genes from Actinomycetes.
[123] Spinocyn
[124] [1141 DowElanco's PCT Publication No. 99/46387.
[125] Spiramycin
[126] [115] US Patent No. of Lilly Corporation 5,098,837
[127] [116] Activator Gene
[128] US Patent No. of Lilly 5,514,544.
[129] Tylosin
[130] [117] Lilly's US Patent No. 5,876,991; U.S. Patent No. 5,672,497; U.S. Patent No. 5,149,638; EP Publication No. 791,655; And EP Publication No. 238,323.
[131] [118] Kuhstoss et al., 1996, Gene 183: 231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.
[132] [119] Tailoring enzymes
[133] Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13 : 349-355. Streptomyces analysis of my access PRA dia new biosynthetic genes of five tiles from the genome of tylBA band (Streptomyces fradiae) on.
[134] The genes may be used in the recombinant DNA expression vector of the present invention regardless of the presence or absence of the gene for polyketide modification. In addition, host cells of the invention can also be made by transforming with a plurality of vectors, each containing a portion of the desired PKS and a modified enzyme gene cluster: US Pat. See 6,033,883. Incorporated herein by this reference.
[135] [121] In order to enhance polyketide production in host cells of the present invention, including Myxococcus , the cells may be transformed to express heterologous phosphopanthetinyl transferases. PKS proteins require phosphopantetheinylation of the ACP domain of the loading and extender modules as well as the PCP domain of any NRPS. Phosphopanthetenylation is mediated by an enzyme called phosphopanthetheinyl transferase (PPTases). To produce functional PKS enzymes in host cells that do not naturally express PPTase capable of acting on the desired PKS enzyme, or to increase the amount of functional PKS enzyme in PPTase-limited host cells, including Sfp (but Heterologous PPTases can be introduced (see PCT Publication Nos. 97/13845 and 98/27203, No. US Pat. No. 6,033,883, incorporated herein by reference). Another suitable PPTase that can be used for this purpose is MtaA from Stigmatella aurantiaca .
[136] [122] Another method provided by the present invention for enhancing polyketide production in any kind of organism, including myxococcus, streptomyces, and solanzium host cells, is directed to streptomycin, rifampicin and / or gentamicin. It is to select cells resistant to. In a preferred embodiment, the host cell producing the polyketide is continuously challenged with each of these compounds (or similar in structure) to isolate the resistant cells with increased polyketide production capacity and select them for selection. Use for steps. In this way, without limitation, it is possible to obtain a high concentration of epothilone or epothilone derivatives and to obtain resistance to streptomycin, rifampicin and gentamicin, such as Myxococcus xanthus host cells.
[137] The host cell of the present invention can be used not only to produce polyketides found in nature but also to produce polyketides produced by the products of recombinant PKS genes and modification enzymes. In one important embodiment, the present invention provides a recombinant DNA expression vector comprising a hybrid PKS. For the purposes of the present invention, a hybrid PKS may comprise all or part of one or more extender modules, loading modules, and thioesterase / cyclase domains of the first PKS and extender modules, loading modules, and thioes of the second PKS. Recombinant PKS comprising all or part of the terase / cyclase domain.
[138] One skilled in the art will recognize that not all or part of the first or second PKS in the hybrid PKS of the present invention need to be separated from a naturally occurring source. For example, very small amounts of AT domains determine their specificity. PCT Disclosure No. See 00/001838 (incorporated herein). With conventional NA synthesis techniques, those skilled in the art can produce de novo DNA compounds of sufficient size to construct useful portions of PKS modules or domains. For the purposes of the present invention, such synthetic DNA compounds are considered to be part of PKS.
[139] As the table shows, there are a variety of PKS genes that serve as readily obtainable DNA sources and sequence information for use in preparing the hybrid PKS-encoding DNA compounds of the present invention. Methods for preparing hybrid PKS-encoding DNA compounds are described in US Pat. 6,022,731; 5,962,290; 5,672,290; 5,672,491; And 5,712,146, and PCT Publication Nos. 98/49315; 99/61599; And 00/047724, each of which is incorporated herein by reference. The hybrid PKS-encoding DNA compound of the invention may be a hybrid of more than two PKS genes. Even when only two genes are used, there are often two or more modules in a hybrid gene in which all or part of the module is derived from a second PKS gene. Those skilled in the art will appreciate that the hybrid PKS of the present invention includes but is not limited to any of the following types of PKS: (i) PKS containing modules in which at least one domain is a heterogeneous module; (ii) PKS containing modules from heterologous PKS; (iii) PKS containing proteins from heterologous PKS; And (iv) a combination of the foregoing.
[140] [126] Hybrid PKS enzymes of the invention are often prepared by substituting the coding sequence of one or more domains of a module from a first PKS with the coding sequence of one or more domains of a module from a second PKS to produce a recombinant coding sequence. do. In general, all references in the text for inserting or substituting KR, DH, and / or ER domains refer to the module's associated KR, DH or ER domain to the corresponding domain from the module from which the domain inserted or substituted is obtained. It includes replacing. If desired or advantageous, the KS and / or ACP of any module may also be replaced with another KS and / or ACP. For example, if the production of the epothilone derivative compound is poor due to the change of the module, productivity can be improved by changing the KS and / or ACP domains of successive modules. For each of these substitutions or insertions, the heterologous KS, AT, DH, KR, ER or ACP coding sequences can be derived from chemical synthesis to obtain another module or hybrid PKS coding sequence of the same or different PKS.
[141] While important embodiments of the present invention relate to hybrid PKS genes, the present invention also recombines without a second PKS gene sequence but differs by one or more mutations and / or deletions from naturally occurring PKS genes. PKS genes are also provided. This deletion may encompass one or more modules or domains and / or be limited to deletions within one or more modules or domains. If the deletion encompasses the entirety of the extender module (except for the NRPS module), the resulting polyketide derivative is at least 2 carbon shorter than the compound produced from the PKS from which the deleted version was derived. Deletion can also encompass NRPS modules and / or loading modules. If the deletion is within one module, the deletion is only one domain, typically a KR, DH or ER domain, or two or more domains, such as both DH and ER domains, or both KR and DH domains, or KR, DH and ER It may also include all three of the domains. The domain of PKS may be functionally "deleted" by mutations such as by random or site-specific mutations. Thus, as illustrated herein, KR domains can be rendered non-functional or not fully functional by mutation. In addition, the specificity of the AT domain can also be altered by mutations such as by random or site-specific mutations.
[142] [128] In order to construct any PKS of the present invention, for example, PCT Publication No. 98/27203, US Patent No. As described in 6,033,883 (all incorporated herein by reference), several different genes of PKS and one or more genes of one or more polyketide modifying enzymes, if necessary, are divided into two or more, often three segments, each segment Can be used to locate the isolated expression vector (PCT Publication No. 00/053361, both of which are incorporated herein by reference). In this way, the assembly and manipulation of the gene for heterologous expression can be made much easier, and each segment of the gene can be altered and the various modified segments can be combined in a single host cell to produce the recombinant PKS gene of the present invention. Can provide. This technique makes it possible to more efficiently build recombinant PKS genes, vectors for expressing these genes, and large libraries of host cells containing these vectors. In this and other contexts, the genes encoding the desired PKS may not only be present in two or more vectors, but may also be ordered or arranged differently from those present in the natural producer organism from which the gene is derived.
[143] [129] In a preferred and exemplary embodiment, the recombinant host cell of the present invention produces an epothilone or an epothilone derivative. Naturally occurring epothilones (including epothilones A, B, C, D, E, and F) and structurally related non-naturally occurring compounds (epothilone derivatives or analogs) are potent cytopathic agents specific for eukaryotic cells . These compounds have utility in treating antifungal agents, chemotherapeutic agents for the treatment of cancer, and immunosuppressants, and in general for the treatment of inflammation or any hyperproliferative diseases such as psoriasis, multiple sclerosis, atherosclerosis and stents. Epothilones are produced only naturally in extremely low concentrations in Sorangium cellulosum cells, to which they have been identified. In addition, Sorangium cellulosum grows very slowly, and Sorangium cellulosum strains are difficult and time-consuming to ferment. One important advantage provided by the present invention is the ability to easily produce epothilones or epothilone derivatives in host cells other than Sorangium cellulosum . Another advantage of the present invention is that the epothilones can be produced in higher concentrations and in higher amounts in the recombinant host cells provided herein than is possible in naturally occurring epothilone producer cells. Another advantage of the present invention is that it is possible to prepare epothilone derivatives in recombinant host cells. Accordingly, the present invention provides recombinant host cells that produce the desired epothilones or epothilone derivatives. In a preferred embodiment, such host cells produce epothilones or epothilone derivatives at a concentration of at least 10 mg / L. In one embodiment, the invention provides host cells that produce one or more epothilones or epothilone derivatives at higher concentrations than are produced in naturally occurring organisms producing epothilones. In another embodiment, the present invention provides host cells that produce a mixture of epothilones that are less complex than the mixture produced by naturally occurring host cells that produce epothilones.
[144] [130] In a particularly preferred embodiment, the host cell of the present invention produces a less complex mixture than naturally occurring cells producing epothilones. As an example, certain host cells of the present invention can produce epothilone D in a less complex mixture than is produced by naturally occurring Sorangium cellulosum , where epothilone D is the main product in the former. And in the latter it is a byproduct. The naturally occurring Sorangium cellulosum producing epothilones generally produces a mixture of epothilones A, B, C, D, E, F and other very small amounts of product, where only epothilones A and B It only exists as the main product. Table 1 below summarizes the epothilones produced in various exemplary host cells of the present invention.
[145] Cell types Produced Epothilones Unproduced Epothilones * One A, B, C, D E, F 2 A, C B, D, E, F 3 B, D A, C, E, F 4 A, B C, D 5 C, D A, B 6 B A, C, D, E, F 7 C A, B, C, E, F
[146] * Or produced only in small quantities
[147] Thus, the recombinant host cell of the present invention also includes a host cell that produces only one desired epothilone or epothilone derivative as a main product.
[148] [132] Based only on the results of domain analysis of epothilone PKS, it can be predicted that the PKS enzyme can catalyze the production of epothilones, optionally labeled "G" and "H". These structures are shown below:
[149]
[150] [133] These compounds differ only in that for epothilone G has hydrogen at C-12, while epothilone H has a methyl group at that position. The diversity of the C-12 positions allows the corresponding AT domain of PKS (extender module 4) to bind to malonyl CoA (which in this case becomes hydrogen), or to methylmalonyl CoA (which is methyl in this case). It is assumed that it is because it has the ability to. However, epothilones G and H have not been observed in nature or in recombinant host cells of the invention. Rather, it is believed that the products of PKS are Epothilones C and D, which differ from Epothilones G and H, respectively, in that they have C-12-C-13 double bonds and lack C-13 hydroxy substituents. Based on the expression of the epothilone PKS genes in heterologous host cells and the products produced by genetic alterations of these genes (described in more detail below), C12-C13 double bonds are formed at epothilones C and D. Dehydration is believed to be carried out by epothilone PKS itself. Epothilones A and B are formed by epoxidation of C12-C13 double bonds with epoK gene products from epothilones C and D, respectively. As described in detail below, epothilones E and F are inserted from hydroxymalonyl CoA instead of malonyl CoA by hydroxylation of C-21 methyl groups or loading modules of epothilone PKS from epothilones A and B, respectively. It can be formed by.
[151] Accordingly, expression of the epoK gene and the epothilone PKS gene in the host cell of the present invention results in the production of epothilones A, B, C and D. If the epoK gene is absent or inactivated or partially inactivated by mutation, epothilones C and D are produced as the main product. When the AT domain of extender module 4 is replaced by an AT domain specific for malonyl CoA, epothilones A and C are produced, and in the absence of a functional epoK gene, epothilone C is produced as the main product. If the AT domain of extender module 4 is replaced by an AT domain specific for methylmalonyl CoA, epothilones B and D are produced as the main product, and in the absence of a functional epoK gene, epothilone D is produced as the main product.
[152] [135] Epothilone PKS and a modified enzyme gene were cloned from Sorangium cellulosum SMP44, an epothilone producing strain. Total DNA was prepared from this strain using the method described by Jaoua et al., 1992, Plasmid 28 : 157-165, herein incorporated by reference. Cosmid libraries were prepared from genomic DNA of Sorangium cellulosum in pSupercos (Stratagene). PCT Publication No. referenced in the text. As described in 00/031237, four overlapping cosmid clones (by the Budapest Treaty, February 17, 1999. American Type Culure Collection (ATCC), 10801, University Blve., Manassas, VA, 20110-2209, USA Deposited with the following accession numbers: pKOS35-70.1A2 (ATCC 203782), pKOS35-70.4 (ATCC 203781), pKOS3570.8A3 (ATCC 203783), and pKOS35-79.85 (ATCC 203780) The whole PKS and modifying enzyme gene clusters were separated from and DNA sequences were determined. DNA sequence analysis revealed a PKS gene cluster with a loading module and nine extender modules. Downstream of the PKS sequence is an open reading frame (ORF) named epoK , which shows strong homology with the cytokine p450 oxidase gene and encodes an epothilone epoxidase modifying enzyme.
[153] The PKS gene is organized into six ORFs. At the polypeptide level, loading module and extender module 1 (NRPS), 2 and 9 represent individual polypeptides; Their corresponding genes are called epoA, epoB, epoC and epoF , respectively. Modules 3, 4, 5 and 6 is that the gene is contained on a single polypeptide, referred to epoD, and modules 7, 9 are contained on another polypeptide whose gene, referred to as epoE. The spacing between ORFs suggests that epoC, epoD, epoE and epoF constitute operons. The epoA, epoB and epoK genes can also form part of this large operon, but there are about 100 bp between epoB and epoC, and 115 bp between epoF and epoK, and if so, a promoter can be contained. Epothilone PKS gene clotor is shown schematically in Scheme 1 below.
[154]
[155] [137] The P450 epoxidase gene, just downstream of epoK, is ORF1, which encodes a polypeptide that may be involved in membrane spanning domains and epothilone delivery. This ORF involves a number of ORFs, including genes that can encode proteins involved in delivery and regulation. A careful examination of this module showed that the epothilones biosynthesis is consistent with their organization and composition. The following description is given at the polypeptide level. The sequences of the AT domains in the loading modules and extender modules 3, 4, 5, and 9 are found in the loading modules as well as C-14, C-12 (epothilones A and C), C-10, and C-2, respectively. Consistent with the consensus sequence for the malonyl loading module, consistent with the presence of the H side chain. The AT domains of modules 2, 6, 7, and 8 resemble the consensus sequence of the methylmalonyl directed AT domain, which in turn resembles the presence of methyl side chains at C-16, C-8, C-6 and C-4. Each match.
[156] [138] The loading module usually contains a KS domain whose residue at the active site is tyrosine, not cysteine. This domain is designated as KS ^Y and serves as a decarboxylase, a kind of its normal function, but cannot function as a condensing enzyme. Thus, the loading module is expected to load malonyl CoA, transfer it to ACP, and decarboxylate to produce the acetyl residues required for condensation by cysteine. Extend module 1 is a non-ribosomal peptide synthetase that activates cysteine and catalyzes condensation with acetate on the loading module. This sequence contains segments very similar to the ATP-binding and ATPase domains required for the activation of amino acids, phosphopanthethenylation sites, oxidation domains, cyclizing domains, and elongatin domains. Extender module 2 determines the structure of the epothilones at C-15-C-17. The presence of the DH domain in module 2 results in the C-16-17 dehydro portion in the molecule. The domain of module 3 is consistent with the epothilone structure at C-14 and C-15; OH, brought about by the action of KR, is used for lactonation of molecules. Extender module 4 controls the structures at C-12 and C-13 where sms double bonds are found in epothilones C and D. Although the sequence of the AT domain appears to resemble those that specify malonate loading, it may also be loaded with methylmalonate, and thus part of the description of the mixture of epothilones found in fermentation broths of naturally occurring organisms. Do it.
[157] [139] A meaningful starting point for the array of functions expected in extender module 4 was detected. This module was expected to direct the synthesis of epothilones C and D as the product of PKS by including the DH domain. The analysis revealed that the space between the AT and KR domains of Module 4 was not large enough to accommodate the functional DH domains. Thus, the degree of reduction in module 4 does not seem to proceed beyond the keto reduction of beta-keto formed after condensation directed by extender module 4. As shown here, the epothilone PKS gene alone is sufficient to impart the ability of producing epothilones C and D to the fast-growing cells of the present invention. Heterogeneous production of epothilones C and D demonstrates the dehydratase function of introducing double bonds. Based on the heterologous expression of the epothilone PKS gene and the product produced by the altered epothilone PKS gene, the dehydration reaction to form this double bond is caused by the DH domain of extender module 5 of the epothilone PKS and the ER domain of module 5 It is believed to be mediated by the production of conjugated diene precursors prior to reduction.
[158] [140] Extender modules 5 and 6 have a complete set of reducing domains (KR, DH and ER) which produce methylene functional groups at C-11 and C-9, respectively. Extender modules 7 and 9 have a KR domain and therefore have hydroxyls in C-7 and C-3, and extender module 8 does not have a functional KR domain, which is consistent with the presence of a keto group in C-5. Extender module 9 also has a thioesterase domain that terminates polyketide synthesis and catalyzes ring closure.
[159] The genes, proteins, modules and domains of epothilone PKS are summarized in Table 2 below.
[160] gene protein module Existing Domain epoAepoAroad KS ^Y mAT Er ACP epoBepoBOne NRPS, condensation, heterocyclizationAdenylation, thionylation, PCP epoCepoC2 KSmmAT DH KR ACP epoDepoD3-6 KS mAT KR ACP; KS mAT KR ACP; KSmAT DH ER KR ACP; KS mmAT DH ERKR ACP epoEepoE7-8 KS mmAT KR ACP; KS mmAT MT DH * KR * AC epoFepoF9 KS mAT KR DH * ER * ACP TE
[161] NRPS-non-rebosomal peptide synthetase; KS-ketosynthase; mAT-malonyl CoA specifying acyltransferase; mmAT-methylmalonyl CoA specifying acyltransferase; DH-dehydratase; ER-enoylreductase; KR-ketoreductase; MT-methyltransferase; TE thioesterase; *-Inactive domains.
[162] [142] Examination of the sequences revealed a translational coupling between epoA and epoB (loading module and extender module 1 NRPS) and between epoC and epoD . Very small gaps were seen between epoD and epoE and epoE and epoF , but more than 100 bp were found between epoB and epoC and e poF and epoK . It may contain such intergenic region promoters.
[163] [143] Thus, epothilone PKS is a multiprotein complex composed of gene products of the epoA, epoB , epoC, epoD , epoE, and epoF genes. To confer host cells with the ability to produce epothilones, the host cells are provided with the recombinant epoA, epoB , epoC, epoD , epoE, and epoF genes of the present invention and any other genes expressible in the host cell, such as epoK . do. Those skilled in the art will appreciate that although epothilones and other PKS enzymes are inferred herein as a single entity, these enzymes are generally multisubunit proteins. Thus, by altering one or more genes encoding one or more of the proteins that make up PKS, inducible PKS (different by deletion or mutation of naturally occurring PKS) or hybrid PKS (consisting of parts of two different PKS enzymes) PKS).
[164] [144] Post-PKS modification or alignment of epothilones consists of multiple steps mediated by a number of enzymes. These enzymes are called tailorgin enzymes or modification enzymes in the text. Expression of the epothilone PKS genes epoA, epoB, epoC, epoD, epoE, and epoF in host cells of the present invention that do not express epoK resulted in C-12-C-13 epoxide of Epothilones A and B instead of C Epothilones C and D with -12-C-13 double bonds are produced. Thus, epothilones C and D are converted to epothilones A and B by epoxidase encoded by the epoK gene. Epothilones A and B are designated as Epothilone E and Epothilone F by the hydroxylase gene, which can be encoded by a gene associated with the Epothilone PKS gene cluster or by another gene of endogenous of Sorranium cellulose . Can be switched. On the other hand, these compounds may be formed by loading modules that bind to starter units other than malonyl CoA (such as hydroxymalonyl CoA). Thus, providing a host cell with one or more recombinant modifying enzyme genes provided by the present invention or using a host cell that naturally expresses (or does not express) a modifying enzyme and / or provides a starter unit other than malonyl CoA. It is thereby possible to produce modified epothilones or epothilone derivatives as necessary.
[165] Accordingly, the present invention provides a wide variety of recombinant DNA compounds and host cells for expressing naturally occurring epothilones A, B, C, and D and derivatives thereof. The present invention also provides recombinant host cells that produce epothilone derivatives modified in a similar manner to epothilones E and F. In addition, any epothilones or epothilone derivatives of the present invention are described in PCT Publication No. According to the method described in 00/039276, it can be converted to the corresponding epothilone E or F derivative.
[166] [146] The present invention also provides a wide range of recombinant DNA compounds and host cells that produce epothilone derivatives. As used herein, the expression epothilone derivative refers to a domain in which at least one domain has been inserted or has been inactivated by deletion or mutation, which has been mutated to alter its catalytic function, or which has a different function. It refers to a compound produced by substituted recombinant epothilone PKS. In any case, the epothilone derivative PKS thus produced functions to produce compounds that differ in structure from naturally occurring epothilones selected from epothilones A, B, C, D, E and F. To aid in understanding the recombinant DNA compounds and host cells provided by the present invention, a detailed description of each module and loading module of epothilone PKS, and novel recombinant derivatives thereof, is provided below.
[167] [147] The loading module of epothilone PKS includes an "inactive" KS domain called KS ^Y , which is due to the presence of tyrosine (Y) residues instead of cysteine residues found in "active" KS domains. It does not perform the condensation reaction mediated by. The KS ^Y domain performs a decarboxylation reaction mediated by the KS domain. Such "inactive" KS domains are usually found in other PKS enzymes with glutamine (Q) residues instead of the cysteine of the active site, which is called the KS ^Q domain. The KS ^Q domains in the fatty acid synthase of rats did not condense but showed a second order of magnitude increase in the decarboxylation reaction. Witkowski et al., 7 Sep. 1999, Biochem. 38 (36): 11643-11650, referenced herein. KS ^Q domain to randomly more effective for decarboxylation than KS ^Y domain, in the case to replace the KS ^Y domain of the epothilone PKS with KS ^Q domain to which any of the epothilone biosynthetic efficiency of a host cell is increased under certain culture conditions Could be This can be done simply by changing tyrosine codons to glutamine codons as described in Example 6 below. This can also be done by replacing the KS ^Y domain with the KS ^Q domain of another PKS such as oleandolide PKS or narbonolide PKS (oleandomycin, narbomycin and pyromycin PKS). And literature in the above table relating to modified enzymes.
[168] [148] The epothilone loading module also contains an AT domain specific to malonyl CoA (believed to be decarboxylated by the KS ^Y domain to produce acetyl groups) and an ACP domain. The present invention provides a recombinant epothilone derivative loading module or coding DNA sequence thereof wherein the malonyl specific AT domain or coding sequence thereof has been modified to have other specificities such as, for example, methylmalonyl CoA, ethylmalonyl CoA, and 2-hydroxymalonyl CoA. When expressed by other proteins of epothilone PKS, this loading module causes the epothilone to be produced in which the methyl substituents of the thiazole ring of the epothilone are replaced with ethyl, propyl and hydroxymethyl, respectively. The present invention provides recombinant PKS enzymes comprising such loading modules and host cells producing such enzymes and polyketides produced thereby. If the AT domain is altered to specify 2-hydroxymalonyl CoA, the corresponding epothilone PKS derivative will produce epothilone E and F derivatives. AT domains specific for 2-hydroxymalonyl CoA will result in polyketides having hydroxyl groups at the corresponding positions in the produced polyketides; The hydroxyl groups can be methylated to produce methoxy groups by polyketide modifying enzymes. See, eg, literature related to FK-520 PKS in the table above. In the end, PKS with 2-hydroxymalonyl specific for the AT domain here refers likewise to polyketides produced by PKS having hydroxyl or methoxy groups at corresponding positions in the polyketide.
[169] [149] The loading module of epothilone PKS also includes an ER domain. This ER domain may be involved in forming one of the double bonds in the thiazole portion of the epothilone (in the reverse reaction of its normal reaction), but may be non-functional. In any case, the present invention is not only a hybrid loading module that contains an ER domain from another PKS (whether active or inactive, with or without KR and DH domains), but instead of the ER domain of the epothilone loading module. Provided are recombinant DNA compounds encoding the epothilone PKS loading module, with or without bands. The present invention also provides recombinant PKS enzymes comprising such loading modules and host cells producing such enzymes and polyketides produced thereby.
[170] The loading module of epothilone PKS may also be replaced by a loading module from heterologous PKS to form a hybrid PKS that makes an epothilone derivative. In one embodiment, the loading module of epothilone PKS is replaced with NRPS as described in the Examples below.
[171] The loading module of epothilone PKS involves the first extender module of PKS, an extender NRPS module specific for cysteine. This NRPS module is naturally expressed as discrete proteind, the product of the epoB gene. In one embodiment, a portion of the NRPS module coding sequence is used in conjunction with a heterologous coding sequence. In this embodiment, the present invention provides for changing the specificity of the NRPS module of epothilone PKS, for example from cysteine to another amino acid. This change is achieved by constructing a coding sequence in which all or part of the epothilone PKS NRPS module coding sequence has been replaced by coding an NRPS module with different specificity.
[172] In one exemplary embodiment, the specificity of the epothilone NRPS module is changed from cysteine to serine or threonine. The modified NRPS module is expressed by another protein of epothilone PKS, and recombinant PKS produces an epothilone derivative wherein the thiazole portion of the epothilone (or epothilone derivative) is changed to an oxazole or 5-methyloxazole portion, respectively. Done. Thus, in an exemplary embodiment, the present invention relates to a host cell, vector and recombinant epo, wherein the adenylated domain of epothilone NRPS is replaced with the adenylated domain of NRPS encoded by the entF gene (which represents serine). Provides Tyrone PKS enzyme. In another exemplary embodiment, the invention relates to host cells, vectors and recombination wherein the NRPS domain has been altered by replacing the adenylated domain of epothilone NRPS with the adenylated domain of NRPS encoded by the bivF gene (which represents threonine). Epothilone PKS enzyme is provided. In one embodiment, substitutions of these NRPS are epothilones that also contain extender module 2, which binds to malonyl CoA instead of methylmalonyl CoA to produce 16-desmethyl derivatives of oxazole and methyloxazole epothilone derivatives. Made in PKS
[173] Meanwhile, the present invention provides a recombinant PKS enzyme consisting of the products of the epoA, epoC, epoD, epoE and epoF genes (or modified versions thereof), with or without NRPS modules from heterologous PKS. Heterologous NRPS modules can be expressed as fusion proteins with epoA or epoC genes, or as discrete proteins. In substituting one module of the PKS for another, one must maintain or use a compatible intermodular linker sequence. PCT Disclosure No. See 00/047724.
[174] In another embodiment, the present invention provides a recombinant epothilone PKS enzyme and a corresponding recombinant DNA compound and vector in which the NRPS module is inactivated or deleted. Thus, the inactive NRPS module protein and coding sequence provided by the present invention have been inactivated by deleting all or part of the PCP domain or by altering the serine of the active site (phosphopanthetinylation site) to another amino acid such as alanine or And those in which adenylated domains have been deleted or inactivated. In any case, the resulting epothilone PKS can function only if a substrate is provided that binds to the KS domain of extender module 2 (or subsequent module) of PKS of the epothilone PKS or the epothilone derivative. In the methods provided by the present invention, so modified cells are preferably injected with activated acylthioesters which are bound by a second, but potentially any subsequent extender module and processed into novel epothilone derivatives. Activated acylthioesters are injected into the host cell to produce the novel epothilone derivatives of the present invention. Epothilone derivatives are prepared by injecting or supplying N-acylcysteamine thioesters (NACS) of novel precursor molecules to host cells expressing PKS or acellular extracts containing them. PCT Disclosure Nos. See US99 / 03986 and 00/044717, both of which are incorporated by reference in Examples 9 and 10 below.
[175] [155] The second (first non-NRPS) extender module of epothilone PKS includes AT, DH, KR and ACP specific for KS, methylmalonyl CoA. The second extender module of epothilone PKS is produced as a discrete protein by the epoC gene. Hybrid modules can be created by using all or part of the second extender module coding sequence in conjunction with other PKS coding sequences. In this embodiment, the present invention is directed to, for example, replacing methylmalonyl CoA specific AT with malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; Deletion of either or both of DH and KR; Substitution of DH or KR or both with DH or KR or both designating different stereochemistry; And / or inserting the ER. The resulting heterologous second extender module coding sequence can be expressed with other proteins that make up PKS synthesizing epothilones, epothilone derivatives or other polyketides. On the other hand, the second extender module of epothilone PKS may be substituted or deleted by a module from heterologous PKS, which may be expressed as a fusion protein or as a discrete protein fused to either the epoB or epoD gene product.
[176] [156] Exemplary recombinant PKS genes of the present invention include alteration of the AT domain coding sequence of the second extender module of epothilone PKS such that the encoded AT domain is converted from methylmalonyl specific AT to malonyl specific AT. Or a substituted one. Such malonyl specific AT domain coding nucleic acids include, but are not limited to, narbnolide PKS, sorafen PKS, rapamycin PKS (ie extender modules 2 and 12), and FK-520 PKS (ie extender module 3). , 7, and 8) can be isolated from the PKS gene encoding. When such a hybrid second extender module is expressed with other proteins that make up the epothilone PKS, the resulting epothilone derivative produced is 16-desmethyl epothilone. In one embodiment, the hybrido PKS also contains a methylmalonyl CoA specific AT domain in extender module 4, and in host cells lacking the functional epoK gene, the resulting compound is 16-desmethyl epothilone D. To be expressed. In another embodiment, hybrid PKS also contains altered NRPS specific for threonine, whereby 5-methyloxazole-16-desmethylepothilone is produced.
[177] In addition, the present invention provides a DNA compound and vector encoding a recombinant epothilone PKS enzyme, and a second (or subsequent) KS module of the second extender module as inactivated as described in Examples 9 and 10. Or the corresponding recombinant protein deleted. In a preferred embodiment, this inactivation is achieved by changing the codons for the active site cysteine to alanine codons. As in the case of the corresponding variants described above for the NRPS module, the resulting recombinant epothilone PKS enzyme is epothilone or epo, unless a precursor is supplied that can be bound and extended by the remaining domains and modules of the recombinant PKS enzyme It does not produce tyrone derivatives. Exemplary precursor compounds are described in Example 10 below. On the other hand, such precursors can simply be provided to host cells expressing only the epoD, epoE, and epoF genes.
[178] The third extender module of epothilone PKS includes AT, KR and ACP specific for KS, malonyl CoA. The third extender module of epothilone PKS is expressed as the product of the protein epoD gene, which also contains modules 4, 5 and 6. The alterations in any of the extender modules 3 to 6 result in expression of a protein, generally comprising four extender module modes, to produce a recombinant epothilone PKS that produces an epothilone derivative. In one embodiment, a hybrid module is made by using all or part of the third extender module coding sequence with other coding sequences. In this embodiment, the present invention provides for example, to replace malonyl CoA specific AT with methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; Delete the KR; Replacing KR with KR specifying another stereochemistry; And / or insert DH or DH and ER. The resulting heterologous third extender module coding sequence is available with a coding sequence of PKS that synthesizes epothilones, epothilone derivatives or other polyketides.
[179] Exemplary recombinant PKS genes of the present invention include that the AT domain is changed from malonyl-specific AT to methylmalonyl-specific AT by altering or replacing the AT domain coding sequence of the third extender module of epothilone PKS. do. Such methylmalonyl specific AT domain coding nucleic acids can be isolated from, for example and without limitation, PKS gene coding DEBS, Narbonolide PKS, Rapamycin PKS and FK-520 PKS. When co-expressed with the remaining modules and proteins of the epothilone PKS or the epothilone PKS derivative, the recombinant PKS produces the 14-methyl epothilone derivative of the invention.
[180] One skilled in the art will recognize that the KR domain of the third extender module of PKS is responsible for forming hydroxyl groups related to the cyclization of epothilones. Eventually, avoiding the KR domain of the third extender module or interfering with the cyclization by adding DH or DH and ER domains, at different positions than linear molecules, or epothilones A, B, C, D, E and F The cyclized molecule will be produced.
[181] The fourth extender module of epothilone PKS includes AT, KR and ACP which can bind to KS, malonyl CoA or methylmalonyl CoA. In one embodiment, all or part of the fourth extender module coding sequence is used in conjunction with another PKS coding sequence to prepare a hybrid module. In this embodiment, the invention replaces, for example, malonyl CoA and methylmalonyl specific AT with malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT ; Delete the KR; And / or replace KR with KR specifying another stereochemistry; And / or insert DH or DH and ER. The resulting heterologous fourth extender module coding sequence is inserted into the protein subunit of recombinant PKS that synthesizes epothilones, epothilone derivatives or other polyketides. On the other hand, the present invention provides recombinant PKS enzymes for epothilones and epothilone derivatives in which the entire fourth extender module is replaced by a deletion or a module from a heterologous PKS.
[182] In a preferred embodiment, the present invention provides an epothilone modified to encode AT which binds to methylmalonyl CoA but does not bind to malonyl CoA (or binds to malonyl CoA but not to methylmalonyl CoA). Provided is a recombinant DNA compound comprising the coding sequence for the fourth extender module of PKS. In one embodiment, such specificity alteration is achieved by mutating the coding sequence for the extender module 4 AT domain. Such mutations can be achieved randomly using mutagens such as UV light or by site-specific mutations. In another embodiment, this alteration of specificity is achieved by replacing all or part of the extender module 4 AT domain coding sequence with the coding sequence of the heterologous AT domain. Accordingly, the present invention provides recombinant DNA compounds and expression vectors and corresponding recombinant PKSs incorporating a hybrid of a fourth extender module and methylmalonyl specific AT. The methylmalonyl specific AT coding sequence can be derived from, for example, without limitation, the coding sequence of oleandolide PKS, DEBS, narbonolide PKS, rapamycin PKS or any other PKS comprising a methylmalonyl specific AT domain. have.
[183] [163] In accordance with the present invention, a hybrid fourth extender module expressed from this coding sequence is typically an extender module 3, 5, and 6 as well as a modified fourth extender module (any one or more of which is optionally epothilones). Derivative epoD gene products, including derivatives of PKS, may be inserted into epothilone PKS (or PKS of epothilone derivatives). Thus, the recombinant methylmalonyl specific epothilone fourth extender module coding sequence provided by the present invention provides another method of producing a desired epothilone compound in a host cell. In particular, these compounds will be epothilones D, B and F, in particular the production of epothilone B depends on whether a functional epoK gene or derivative thereof is present.
[184] [164] The present invention also provides a recombinant DNA compound comprising the coding sequence of the fourth extender module of epothilone PKS modified to encode AT which binds to malonyl CoA but not to methylmalonyl CoA. The present invention provides a corresponding recombinant PKS in which the recombinant DNA compound and the vector and the hybrid fourth extender module are inserted into the derivative epoD gene product. When inserted into epothilone PKS (or PKS of an epothilone derivative), the resulting recombinant epothilone PKS produces epothilones C, A and E, wherein the production of epothilone A depends on whether or not a functional epoK gene is present. Depends.
[185] [165] In another embodiment, the invention provides a recombinant host cell producing 12-desmethyl-12-ethyl-epothilone D. In this embodiment, the present invention provides host cells in which the AT domain of extender module 4 expresses a recombinant epothilone PKS derivative substituted by an ethylmalonyl CoA-specific extender module, such as from an FK520 or nidamycin PKS enzyme. . In one embodiment, the host cell is a recombinant host cell that has increased productivity of ethylmalonyl CoA by expressing crotonyl CoA reductase encoded under the control of a heterologous promoter or by a gene from a heterologous host cell ( ccr gene). . In one embodiment, the host cell is a myxococcal host cell that expresses the ccr gene isolated from Streptomyces host cell. In another embodiment, the host cell is modified to express or overexpress E. coli atoA, D, and E genes that deliver butyrate and convert it to butyryl CoA which is converted to ethylmalonyl CoA.
[186] [166] In addition to substituting the endogenous AT coding sequence with the coding sequence of AT specific for methylmalonyl CoA, KR domain coding sequences can be substituted for other KR, DH and KR (eg, non-limiting examples of rapamycin PKS). Module 10 or derived from modules 1 or 5 of FK-520 PKA), or coding sequences of DH, KR and ER. If KR is replaced with another KR or with KR and DH and no change is made to extender module 5 (or other portion of PKS), recombinant epothilone PKS produces epothilones C and D, which are DH of extender module 5 This is because the domain mediates the formation of C-12-C-13 double bonds in epothilones C and D. Recombinant epothilone PKS produces 12,13-dihydro-epothilones C and D if KR is replaced with KR, DH and ER and no changes are made to extender module 5 (or other site of PKS). In addition, if KR is replaced with inactive KR or KR is inactivated, recombinant epothilone PKS produces 13-oxo-11,12-dehydro-epothilones C and D.
[187] Thus, the present invention provides a recombinant epothilone PKS in which the KR domain of extender module 4 is inactivated by substitution by a nonfunctional KR domain from a mutation, deletion or other PKS. This recombinant PKS mainly produces 13-oxo-11,12-dehydro epothilone B; Among the compounds produced by this microorganism, the C-11-C-12 double bonds observed are double bonds formed in the polyketide chain beginning to grow by the DH domain of extender module 5 prior to reduction by the ER domain of the module. It is believed to be derived from the migration of. The invention also provides host cells that produce this novel polyketide. For example, Myxococcus xanthus strain K122-56, which was deposited on November 21, 2000, in the American Type Culture Collection, 10801 University Blvd. Manassas, VA 20110-2209 USA, under the Budapest Treaty. The number PTA-2714 has been assigned to inactivate the KR domain of module 4 by deletion, thereby degrading 13-oxo epothilones A and B and their dehydro derivatives (mainly 13-oxo-11,12-dehydroepothilone B). Contains the epothilone PKS gene.
[188] The fifth extender module of epothilone PKS includes AT, DH, ER, KR and ACP domains that bind KS, malonyl CoA. In one embodiment, a DNA compound comprising a sequence encoding a fifth extender module of epothilone PKS is inserted into a DNA compound comprising a coding sequence of epothilone PKS or recombinant epothilone PKS producing an epothilone derivative. In another embodiment, a portion of the fifth extender module coding sequence is used in conjunction with another PKS coding sequence to make a hybrid module coding sequence and a hybrid module encoded by it. In this embodiment, the invention optionally replaces malonyl CoA specific AT with methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT to specify different stereochemistry; Deleting one, two or all of ER, DH and KR; And / or replacing any one, two or all of ER, DH, and KR with KR, DH and KR, or KR, DH and ER. The resulting hybrid fifth extender module coding sequence can be used with the coding sequence of PKS to synthesize epothilones, epothilone derivatives or other polyketides. On the other hand, the fifth extender module of the epothilone PKS is entirely substituted or deleted by the heterologous PKS module, thereby producing a protein constituting the PKS that produces the epothilone derivative in association with the other protein of the epothilone PKS or a derivative thereof. have.
[189] [169] In an exemplary recombinant PKS gene of the present invention, the AT domain coding sequence of the fifth extender module of epothilone PKS is altered or substituted to change the AT domain encoded so that it becomes methylmalonyl-specific AT from malonyl-specific A. Recombinant epoD gene derivatives are included. Such methylmalonyl specific AT domain coding nucleic acids can be isolated from non-limiting examples, PKS genes encoding DEBS, Narbonolide PKS, Rapamycin PKS and FK-520 PKS. When such recombinant epoD genes are expressed with the epoA, epoB, epoC, epoE, epoF and / or epoK genes (or derivatives thereof), the PKS composed thereof produces 10-methyl epothilones or derivatives thereof. Another recombinant epoD gene provided by the present invention includes not only this modified module 5 coding sequence, but also a module 4 coding sequence encoding an AT domain that binds only to methylmalonyl CoA. Inserting the epoA, epoB, epoC, epoE, epoF and / or epoK genes into the PKS , the recombinant epoD gene derivative product produces 10-methyl epothilone B and / or D derivatives.
[190] Another exemplary recombinant epoD gene derivative of the present invention is the substitution or mutation of one or more ER, DR, and KR domain coding sequences for the fifth extender module of epothilone PKS: (i) nonfunctional ER , DH or KR domains; (ii) only functional KR domains; (iii) only functional KR and DH domains; (iv) those providing functional ER, DH or KR domains from other PKS. The discovery that the DH domain of extender module 5 is responsible for the formation of C-12-C-13 double bonds in epothilones C and D suggests that all organisms, including Soranium cellulose , and the recombination containing the epothilone PKS gene Provided are novel methods of the present invention for producing epothilones and epothilone derivatives in host cells. In addition, it has been found that the DH domain of extender module 6 may also act on the beta-carbonyl of newly formed polyketides bound to the previous module, which are available according to the method of the present invention for making novel epothilone derivatives. I lost.
[191] Thus, if the extender module 5KR, DH and ER domains of the three modus are deleted or inactivated, recombinant epothilone PKS produces 13-hydroxy-11-oxo analogs of epothilones A and B. If the DH and ER domains are deleted or inactivated, recombinant epothilone PKS produces 13-hydroxy-10,11-dehydro-epothilone, mainly 13-hydroxy-10,11-dehydroepothilone D. The present invention also provides a host cell for producing this novel polyketide. For example, Myxococcus xanthus strain K122-148, which was deposited on November 21, 2000, in the American Type Culture Collection, 10801 University Blvd. Manassas, VA 20110-2209 USA, under the Budapest Treaty. Numbered PTA-2711) represents the epothilone PKS gene wherein the Dh, KR and ER domains of extender module 5 are replaced by only the KR domain to produce 13-hydroxy-10,11-dehydro-epothilone D. It contains. The present invention also provides novel epothilone derivatives produced by this strain. If only the ER domain is deleted or otherwise inactivated, recombinant epothilone PKS produces 10,11-dehydro analogs of epothilones C and D, mainly 10,11-dehydro epothilones. Thus, in one aspect, the present invention provides recombinant epothilone PKS wherein the ER domain of extender module 5 is inactivated or deleted by mutation to produce 10,11-dehydro-epothilone D. In another embodiment, the present invention provides a Sorangium cellulosum host cell that produces 10,11-dehydro-epothilone D due to a mutation in the coding sequence of the ER domain of extender module 5 of epothilone PKS. to provide.
[192] [172] These recombinant epoD gene derivatives of the present invention are expressed with the corresponding epoA, epoB, epoC, epoE and epoF genes or otherwise altered recombinant epo genes (and may contain additional modifications of their own) and corresponding epothilone derivatives. Produces PKS to make. For example, one of the recombinant epoD gene derivatives provided by the present invention also includes a module 4 coding sequence encoding an AT domain that binds only to methylmalonyl CoA. As noted above, functionally similar epoD genes that produce epothilone C-11 derivatives may also be made by inactivating any, two or all of the ER, DH and KR domains of the epothilone fifth extender module. Another way to alter this domain in any module is to substitute a complete set of desired domains taken from another module of homologous or heterologous PKS coding sequence. In this way, the original structure of the PKS is maintained. In addition, KR and DH or KR, DH, and ER domains that function together in native PKS are preferably used in recombinant PKS when present. Exemplary alternative domains of the aforementioned substituents include, for example, inactive KR domains from rapamycin PKS extender module 3, KR domains from rapamycin PKS extender module 5 and KR and DH domains from rapamycin PKS extender module 4 However, the present invention is not limited thereto. Other such inactive KR, active KR and active KR and DH domain coding nucleic acids can be isolated from, but are not limited to, the PKS genes encoding DEBS, Narbonolide PKS, and FK-520 PKS, for example. Each of the resulting PKS enzymes is further induced in vitro by standard chemical methodology to produce polyketide compounds capable of producing the semisynthetic epothilone derivatives of the present invention.
[193] The sixth extender module of epothilone PKS includes AT, DH, ER, KR and ACP which bind to KS, methylmalonyl CoA. In one embodiment, a portion of the sixth extender module coding sequence can be used with other PKS coding sequences to form a hybrid module. In this embodiment, the invention optionally includes, for example, replacing methylmalonyl CoA specific AT with malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; Deleting one, two or all of ER, DH and KR; And / or replacing any one, two or all of ER, DH and KR with KR, DH, and KR, or KR, DH and ER. The resulting heterologous sixth extender module coding sequence can be used with the coding sequence of the protein subunit of PKS to make epothilones, epothilone derivatives, or other polyketides. On the other hand, PKS for the epothilone derivatives can be produced by deleting the sixth extender module of epothilone PKS or replacing the whole with a heterologous PKS module.
[194] In an exemplary recombinant PKS gene of the present invention, the AT domain coding sequence of the sixth extender module of epothilone PKS is altered or substituted so that the AT domain encoded thereby is malonyl-specific from methylmalonyl-specific AT. This includes changes to AT. Such malonyl specific AT domain coding nucleic acids can be isolated from, for example and without limitation, the PKS gene encoding Narbonolide PKS, Rapamycin PKS, and FK-520 PKS. When the recombinant epoD gene of the present invention encoding this hybrid module 6 is expressed with other epothilone PKS genes, the recombinant PKS produces an 8-desmethyl epothilone derivative. This recombinant epoD gene derivative may also be further altered to itself to produce PKS that is expressed with an epo gene derivative comprising other alterations or that produces a corresponding 8-desmethyl epothilone derivative. For example, one recombinant epoD gene provided by the present invention also includes a module 4 coding sequence that encodes an AT domain that binds only methylmalonyl CoA. When inserted into PKS along with the epoA, epoB, epoC, epoE and epoF genes, the recombinant epoD gene product results in the production of 8-desmethyl derivatives of epothilone B (if a functional epoK gene is present) and D.
[195] [175] Other exemplary recombinant epoD gene derivatives of the present invention include the ER, DH and KR domain coding sequences of the sixth extender module of epothilone PKS comprising (i) KR and DH domains; (ii) KR domains; And (iii) those substituted by those encoding inactive KR domains. These recombinant epoD gene derivatives of the invention, when expressed with other epothilone PKS genes, correspond to (i) C-9 alkenes, (ii) C-9 hydroxy (both epimers, additional KS in the next module). And / or if no ACP substitution occurs, only one of them can be processed), and (iii) a C-9 keto (C-9-oxo) epothilone derivative. Functionally equivalent sixth extender modules can also be made by inactivating any one, two or all three of the ER, DH and KR domains of the epothilone sixth extender module. For example, the present invention is directed to the Myxococcus xanthus strain K39-164 (the strain is dated Nov. 21, 2000 to the American Type Culture Collection, 10801 University Blvd. Manassas, VA 20110-2209 USA, under the Budapest Treaty). Deposited to be assigned accession number PTA-2711), which contains the epothilone PKS gene in which the KR domain of extender module 6 is inactivated by mutations to produce 9-keto-epothilone D. The present invention also provides novel epothilone derivatives produced by this strain.
[196] Thus, the recombinant epoD gene derivative may be expressed with other recombinant epo gene derivatives including other alterations or may be further altered by itself to produce PKS to produce the corresponding C-9 epothilone derivatives. For example, one recombinant epoD gene derivative provided by the present invention also includes module 4 coding sequences encoding an AT domain that only binds methylmalonyl CoA. When inserted into PKS with epoA, epoB, epoC, epoE and epoF genes, the recombinant epoD gene product produces C-9 derivatives of epothilones B and D, depending on the presence of a functional epoK gene.
[197] Substituted domains by the substituents described above include inert KR domains from rapamycin PKS module 3 for ketone formation, KR domains from rapamycin PKS module 5 for alcohol formation and rapamycin PKS module 4 for alkene formation. KR and DH domains from include, but are not limited to. Such other inactive KR, active KR, and active KR and DH domain coding nucleic acids can be isolated from, but not limited to, the PKS gene encoding DEBS, Narbonolide PKS, and FK-520 PKS. Each of the resulting PKSs is further induced ex vivo by standard chemical methodology to produce a polyketide compound comprising a functional group at the C-9 position capable of producing the semisynthetic epothilone derivative of the present invention.
[198] The seventh extender module of epothilone PKS includes AT, KR and ACP specific for KS, methylmalonyl CoA. The seventh extender module of epothilone PKS is contained in the gene product of the epoE gene, which also contains the eighth extender module. In one embodiment, a DNA compound comprising a sequence encoding a seventh extender module of epothilone PKS is expressed to form a protein that, together with another protein, constitutes PKS producing an epothilone PKS or an epothilone derivative. In these and related embodiments, the seventh and eighth extender modules of epothilone PKS or derivatives thereof are generally a single protein and are expressed with the epoA, epoB, epoC, epoD and epoF genes or derivatives thereof to constitute PKS. In another embodiment, some or all of the seventh extender module coding sequence is used in conjunction with other PKS coding sequences to make a hybrid module. In this embodiment, the present invention comprises, for example, replacing methylmalonyl CoA specific AT with malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; Delete the KR; Replacing KR with KR specifying another stereochemistry; And / or inserting DH or DH and ER. The resulting heterologous seventh extender module coding sequence is optionally used with other coding sequences to express the proteins that make up the PKS that synthesizes the epothilones, the epothilone derivatives, or other polyketides with the other proteins. On the other hand, by replacing or deleting the coding sequence of the seventh extender module in the epoE gene with the sequence for the heterologous module, it can be expressed to make PKS of the epothilone derivative together with the epoA, epoB, epoC, epoD, and epoF genes. Recombinant epoE gene derivatives can be prepared.
[199] [179] An example of a recombinant epoE gene derivative of the present invention is that the AT domain coding sequence for the seventh extender module of epothilone PKS is altered or substituted such that the AT domain encoded is methylmalonyl specific from malonyl specific. Alterations to AT include, but are not limited to, such malonyl-specific AT domain encoding nucleic acids, such as, but not limited to, isolation from PKS genes encoding navonolide PKS, rapamycin PKS, and FK-520 PKS. can do. When expressed with other epothilone PKS genes, epoA, epoB, epoC, epoD and epoF or derivatives thereof, PKS of an epothilone derivative having C-6 hydrogen instead of C-6 methyl is produced. Thus, if this gene does not contain other alterations, the compound produced is 6-desmethyl epothilone.
[200] The eighth extender module of epothilone PKS includes KS, AT, inactive KR and DH domains specific for methylmalonyl CoA, methyltransferase (MT) domain, and ACP. In one embodiment, the DNA compound comprising the sequence encoding the eighth extender module of epothilone PKS is expressed with PKS producing the epothilone derivative or with other proteins making up the epothilone PKS. In yet another embodiment, all or part of the eighth extender module coding sequence is used in conjunction with another PKS coding sequence to make a hybrid module. In this embodiment, the present invention provides, for example, substitution of methylmalonyl CoA specific AT with malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; Delete inert KR and / or inert DH; Replacing inactive KR and / or DH with active KR and / or DH; And / or inserting the ER. The resulting heterologous eighth extender module coding sequence is optionally used in conjunction with another coding sequence to be expressed as a protein constituting the PKS that synthesizes the epothilones, the epothilone derivatives, or other polyketides with the other proteins. On the other hand, by replacing or deleting the coding sequence of the eighth extender module in the epoE gene with the sequence for the heterologous module, it can be expressed to make PKS of the epothilone derivative together with the epoA, epoB, epoC, epoD, and epoF genes. Recombinant epoE gene derivatives can be prepared.
[201] The eighth extender module of epothilone PKS also includes a methylated or methyltransferase (MT) domain having the activity of methylating the epothilone precursor. By deleting this function, at the corresponding C-4 position of the epothilone molecule, the epo lacks one or both methyl groups, depending on whether the AT domain of the eighth extender module has changed its malonyl-specific AT domain The recombinant epoD gene derivatives of the present invention can be produced, which can be expressed by other epothilone PKS genes or derivatives thereof that make tyrone derivatives.
[202] The ninth extender module of epothilone PKS includes KS, malonyl CoA specific AT, KR, inert DH and ACP. The ninth extender module of epothilone PKS is expressed as a protein that is the product of the epoF gene, which also contains the TE domain of epothilone PKS. In one embodiment, a DNA compound comprising a sequence encoding a ninth extender module of epothilone PKS is expressed as a protein along with other proteins to construct a PKS or epothilone PKS that produces an epothilone derivative. In these embodiments, the ninth extender module is generally expressed as a protein that also contains the TE domain of epothilone PKS or heterologous PKS. In another embodiment, some or all of the ninth extender module coding sequence is used in combination with other PKS coding sequences to make a hybrid module. In this embodiment, the present invention comprises, for example, replacing a malonyl CoA specific AT with methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; Delete the KR; Replacing KR with KR specifying another stereochemistry; And / or inserting DH or DH and ER. For example, substituting the AT domain of extender module 9 with a methylmalonyl CoA specific AT domain results in the recombination epothilone PKS that produces 2-methyl-epothilones A, B, C and D in the recombinant myxococcal host cells of the present invention. Caused. The resulting heterogeneous ninth extender module coding sequence is expressed with other proteins that make up PKS synthesizing epothilones, epothilone derivatives, or other polyketides. On the other hand, the present invention provides a PKS of an epothilone derivative in which the epothilone or ninth extender module has been replaced by a module from a heterologous PKS or deleted in its entirety. In the latter example, the TE domain is expressed as a discrete protein or fused to an eighth extender module.
[203] [183] In another embodiment, the invention is directed to a gene encoding a thioesterase type II protein ("TE II"), as well as a heterologous PKS gene cluster (the same kind of unmodified, naturally occurring host cell). A PKS gene cluster) is provided. In a preferred embodiment, the TE II gene is preferably heterologous to the PKS gene cluster—the TE II gene is not derived from the same gene cloister as PKS. As an example, in one embodiment the recombinant host cell of the invention comprises a gene encoding the expression of an epothilone PKS or an epothilone PKS derivative. According to this aspect of the invention, the host cell is modified to contain a TE II gene isolated from a PKS gene cluster other than the epothilone PKS gene cluster. Examples include the pyromycin PKS gene of Streptomyces venezuelae and the tmbA PKS gene cluster of Sorangium cellulosum , which is described in US Patent No. 6,090,601; Aug. 31, 1998. US Patent Application No. 144,085; US Patent Application No. 60 / 271,245, filed February 15, 2001, all of which are incorporated herein by reference.
[204] [184] Examples of recombinant epothilone derivative PKS genes of the invention, identified by listing the modified specificity of a hybrid module (another module with the same specificity as epothilone PKS), include:
[205] (a) module 4 with methylmalonyl specific AT (mmAT) and KR and module 2 with malonyl specific AT (mAT) and KR;
[206] (b) module 4 with mmAT and module 3 with mmAT;
[207] (c) module 4 with mmAT and module 5 with mmAT;
[208] (d) module 4 with mmAT and module 5 with mmAT and only DH and KR;
[209] (e) module 4 with mmAT and module 5 with mmAT and KR only;
[210] (f) module 4 with mmAT and module 5 with mmAT and only inactive KR;
[211] (g) module 4 with mmAT and module 6 with mAT;
[212] (h) module 4 with mmAT and module 6 with mAT and only DH and KR;
[213] (i) module 4 with mmAT and module 6 with mAT and KR only;
[214] (j) module 4 with mmAT and module 6 with mAT and only inactive KR;
[215] (k) module 4 with mmAT and module 7 with mAT;
[216] (l) hybrids (d) to (f) except that module 5 has mAT;
[217] (m) hybrids (h) to (j) except that module 6 has mmAT;
[218] (n) hybrids (a) to (m) except that module 4 has mAT.
[219] The above list is exemplary only and the present invention is not limited thereto. The present invention includes not only two hybrid modules other than those shown, but also enzymes and recombinant epothilone PKS genes having three or more hybrid modules.
[220] [185] The host cells of the present invention can be grown and fermented under conditions known in the art for other purposes to produce the compounds of the present invention. The invention also provides new methods for fermenting the host cells of the invention. The compounds of the present invention can be isolated and purified from the fermentation broth of these cultured cells, for example, according to the method of Example 3 described below.
[221] [186] The present invention provides several methods for fermenting Myxococcus strains for the production of polyketides and other products. Prior to the present invention, Myxococcus had not been fermented for the purpose of producing any polyketide other than TA and sapramycin , which are naturally produced by certain Myxococcus strains. Thus, in one aspect, the present invention is useful, including but not limited to polyketides, nonribosome peptides, epothilones, lipases, proteases, other proteins, lipids, glycolipids, and polyhydroxyalkanoates. It is possible to use Myxococcus as a production host for producing bioactive compounds by fermentation.
[222] [187] Among the methods provided by the present invention, there are methods for producing Myxococcus strains, storage cell banks, and methods for adaptation to fermentation medium. Prior to the present invention, frozen cell banks of Myxococcus strains adapted for production in oil-based fermentation media have never been made, and without such adaptation, in particular, Myxococcus strains in oil-based fermentation media These methods are especially important because they die well.
[223] [188] The invention also provides a useful fermentation medium and the fermentation process of the invention for raising the mikso Rhodococcus (Myxococcus). Surprisingly, mikso Lactococcus glass tooth (Myxococcus xanthus) and other mikso Rhodococcus (Myxococcus) strains do not use a carbohydrate, glycerol, alcohol or the intermediates the TCA cycle as the carbon source. Before the present invention was invented, fermentation of Myxococcus xanthus was carried out in a medium based on protein. However, NH ₄ is increased to concentrations toxic to growth in the protein based medium to limit fermentation. According to the present invention, Myxococcus strains are fermented in a medium containing oil and / or fatty acids as the carbon source.
[224] [189] Examples of oils and fatty acids useful in this method include methyl oleate; Oils derived from coconut, lard, rapeseed, sesame, soybean and sunflower; Salad oil; Self-emulsifying oils such as Agrimul CoS2, R5O5, and R5O3; Glycerol oleate, including glycerol mono oleate and glycerol trioleate; Odd chain esters such as methyl heptadecanoate, methyl nonadecanoate, and methyl pelaronate; Ester chains such as propyl oleate and ethyl oleate; Vegetable methyl oleate; Methyl stearate; Methyl linoleate; Oleic acid; And phosphatidyl choline derived or pure from soybean or egg yolk. Thus, oils derived from any plant or grain, such as sunflower or soybean oil, oil from any animal, such as free or esterified fatty acids of any chain length, saturated or unsaturated, respectively, phosphatidyl choline or Natural or synthetic fatty acid mixtures such as methyl pelargonate, industrial fermented milks such as Cognis Corporation's Agrimul series, and the like can all be used in the present invention. In a preferred embodiment, it is preferred to use methyl oleate as the carbon source in the fermentation medium. In general, oils that are liquid at room temperature are preferred over solid oils, mainly because of their ease of dispersion. Other important constituents of the fermentation medium contain trace metals such as Fe and Cu, which increase productivity and growth in complex and defined media in batch and fed-batch processes. give. Medium containing methyl oleate and trace metals is preferred for producing epothilones.
[225] [190] In one embodiment, the present invention provides a fermentation medium for host cells of the present invention containing no or only a small amount of animal material. Due to the potential for contamination by infectious agents such as viruses and prions, and the use of animal by-products in the fermentation process for the production of compounds to be administered to humans or animals, the use of fermentation media containing no or only small amounts of animal material Would be preferred. Such media are provided for use in the methods of the invention. The oils or fatty acids contained in the fermentation medium may be from non-animal sources such as plants. In addition, animal materials may be replaced with equivalent but not identical materials derived from non-animal sources. For example, casitone, a pancreatic digest of casein, a milk protein, can be replaced with protein hydrolysates from non-animal sources, including but not limited to vegetable sources, such as plant protein hydrolysates.
[226] [191] In general, a fed-batch process is preferred for fermentation. Feeds allow cells to effectively use nutrients (eg, cells break down and metabolize carbon into CO ₂ and H ₂ O instead of generating toxic organic acids). High nutrient concentrations can inhibit secondary metabolism, and if fermentation injects nutrients at rates below the inhibition threshold, productivity can be higher.
[227] [192] The fermentation methods of the present invention also include methods that are particularly relevant for the production of epothilones and fermentation media useful for these methods. In one embodiment, propionate and acetate can be used to influence the ratio of epothilone D: C (or B: A) and the titer of epothilone obtained. This effect is minimal in the preferred methyl oleate / trace metal fermentation medium, while in other media such as CTS medium the effect may be quite significant. Increasing the amount of acetate in the fermentation medium can increase the growth of myxococcus and epothilone production. Acetylone alone can dramatically increase the titer of epothilone C (or epothilone A) and the titer of epothilone D (or epothilone A) can dramatically decrease. Propionate does not increase epothilone titers alone and may reduce titers at high concentrations. However, propionate and acetate together may shift production from epothilone C (or epothilone A) to epothilone D (or epothilone B). One preferred medium for producing epothilone D contains kaziton, 10 mM acetate and 30 mM propionate. Medium containing an odd chain of fatty acids may lower the productivity of epothilone C upon fermentation of myxococcus xanthus cells producing epothilone C and D. Trace metals can also increase the epothilone D production and the epothilone D; C ratio in the presence of acetate and in the absence of oil in the fermentation medium.
[228] [193] The present invention also provides a method for purifying epothilone from a fermentation medium and a method for preparing epitolone in crystalline form. In general, the purification process involves the step of capturing the epothilone on the XAD resin during the fermentation, eluting from the resin, extracting the solid phase, chromatography and then crystallizing. This method is described in detail in Example 3, and for epothilone D, this method is preferably illustrated, but the method includes, but is not limited to, naturally occurring epothilones and epothilone analogs produced by the host cells of the present invention (but It is generally available to prepare crystalline epothilones.
[229] [194] Thus, in another embodiment, the present invention provides novel epothilone derivative compounds in isolated purified form useful in the fields of agriculture, veterinary medicine, and medicine. In one embodiment, this compound is useful as a fungicide. In another embodiment, the compound is useful for chemotherapy of cancer. In another embodiment, the compounds are useful for preventing undesirable cell growth, including, but not limited to, treatment of hyperproliferative diseases such as inflammation, autoimmune diseases and psoriasis, and prevention of cell growth in stents. . In a preferred embodiment, the compound is an epothilone derivative that is at least as strong as epothilone B or D on tumor cells. In another embodiment, this compound is useful for the preparation of another compound. In a preferred embodiment, the compound is formulated in a mixture or solution for administration to humans or animals.
[230] [195] In addition to the epothilones produced by the host cells of the present invention, novel epothilone analogs of the present invention are also described in PCT Patent Publication Nos. 93/10121, 97/19086, 98/08849, 98/22461, 98/25929, 99/01124, 99/02514, 99/07692, 99/27890, 99/39694, 99/40047, 99/42602, 99 / 43320, 99/43653, 99/54318, 99/54319, 99/54330, 99/65913, 99/67252, 99/67253, and 00/00485, and US Pat. Can be induced and formulated as described in 5,969,145.
[231] [196] Compounds of the present invention include, but are not limited to, 14-methyl epothilone derivatives (made using hybrid module 3 of the present invention having AT binding to methylmalonyl CoA instead of malonyl CoA); 8,9-dehydro epothilone derivatives (made using hybrid module 6 of the present invention having DH and KR instead of ER, DH, and KR); 10-methyl epothilone derivatives (made using the hybrid module 5 of the present invention having AT binding to methylmalonyl CoA instead of malonyl CoA); 9-hydroxy epothilone derivatives (made using hybrid module 6 of the present invention having KR instead of ER, DH, and KR); 8-desmethyl-14-methyl epothilone derivative (hybrid module 6 having hybrid module 6 which binds to malonyl CoA instead of methylmalonyl CoA and AT which binds methylmalonyl CoA instead of malonyl CoA) Created); 8-desmethyl-8,9-dehydroepothilone derivative (made by hybrid module 6 of the present invention having AT and KR instead of ER, DH and KR and AT specifying malonyl CoA instead of methylmalonyl CoA) ); And 9-oxo-epothilone D. Other preferred novel epothilones of the present invention include those described in Example 11 and the following.
[232] In one aspect of the invention there is provided a compound represented by the following formula:
[233]
[234] In the formula,
[235] R ¹ , R ² , R ³ , R ⁵ , R ¹¹ , and R ¹² are each independently hydrogen, methyl or ethyl;
[236] R ⁴ , R ⁶ , and R ⁹ are each independently hydrogen, hydroxyl or oxo;
[237] Or R ⁵ and R ⁶ form a carbon carbon double bond;
[238] R ⁷ is hydrogen, methyl, or ethyl;
[239] R ⁸ and R ¹⁰ are both hydrogen or together form a carbon carbon double bond or an epoxide;
[240] Ar is aryl;
[241] W represents O or NR ¹³ , where R ¹³ is hydrogen, C _1-10 aliphatic, aryl or alkylaryl. In another embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R provided that at least one of R ¹ , R ⁴ , R ⁵ , R ⁶ , R ⁹ , and R ¹¹ is not hydrogen. ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and R ¹³ , Ar and W are provided as described above.
[242] In another embodiment, there is provided a compound of Formula (I) having the following definitions:
[243] R ¹ , R ² , R ³ , and R ¹¹ are each independently hydrogen or methyl;
[244] R ⁴ and R ⁹ are each independently hydrogen, hydroxyl or oxo;
[245] R ⁵ and R ⁶ are both modulus hydrogens or together form a carbon carbon double bond;
[246] R ⁷ and R ¹² are both methyl;
[247] Both R ⁸ and R ¹⁰ form hydrogen or a carbon carbon double bond together;
[248] Ar is heteroaryl;
[249] W is O or NR¹³Where R¹³Silver r^One, R⁴, R⁵, R⁶, R⁹, And R¹¹Assuming that at least one of is not hydrogen_1-5 Alkyl or hydrogen).
[250] In another aspect of the present invention, a compound of the following formula is provided:
[251]
[252] Wherein R ⁴ , R ⁶ , and R ⁹ are each independently hydrogen, hydroxyl or oxo;
[253] R ⁵ , R ¹¹ and R ¹² are each independently hydrogen, methyl or ethyl; or
[254] R ⁵ and R ⁶ together form a carbon carbon double bond;
[255] R ⁷ is hydrogen, methyl or ethyl;
[256] R ⁸ and R ¹⁰ both form hydrogen or carbon carbon double bonds or epoxides;
[257] Ar is aryl;
[258] W is O or NR ¹³ , wherein R ¹³ is hydrogen or C _1-5 alkyl. In another embodiment, R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R provided that at least one of R ⁴ , R ⁵ , R ⁶ , and R ⁹ is not hydrogen ^There is also provided a compound of Formula II, wherein ¹¹ , R ¹² , R ¹³ , Ar, and W are as described above.
[259] In another embodiment, compounds of Formula II are provided having the following definitions:
[260] R ⁴ and R ⁹ are each independently hydrogen, hydroxyl or oxo;
[261] R ₅ and R ⁶ are each hydrogen or together form a carbon carbon double bond;
[262] R ⁷ and R ¹² are both methyl;
[263] R ⁸ and R ¹⁰ both form a hydrogen or carbon carbon double bond;
[264] Ar is 2-methyl-1,3-thiazolinyl, 2-methyl-1,3-oxazolinyl, 2-hydroxymethyl-1,3-thiazolinyl, or 2-hydroxymethyl-1,3 Oxazolinyl;
[265] W is NH or O, provided that at least one of R ⁴ , R ⁵ , R ⁶ , and R ⁹ is not hydrogen.
[266] In another embodiment, a compound is provided having the formula:
[267]
[268] In the formula,
[269] R ¹ , R ² , R ³ , R ⁵ , R ¹¹ , R ¹² are each independently hydrogen, methyl or ethyl;
[270] R ⁶ is hydrogen; or
[271] R ⁵ and R ⁶ together form a carbon carbon double bond;
[272] R ⁷ is hydrogen, methyl, or ethyl;
[273] Ar is aryl;
[274] W is O or NR¹³Where R¹³Silver hydrogen or C_1-5 Alkyl). In another embodiment, R^One, R⁵, R⁶, And R¹¹R on the assumption that at least one of is not hydrogen^One, R², R³, R⁵, R⁶, R⁷, R¹¹, R¹²And R¹³Compounds of Formula III are provided, wherein Ar and W are as described above. Compounds of Formula III are provided.
[275] In another embodiment, a compound of Formula III is provided having the following definitions:
[276] R ¹ , R ² , R ³ , and R ¹¹ are each independently hydrogen, methyl or ethyl;
[277] R ⁵ and R ⁶ are each independently hydrogen or together form a carbon carbon double bond;
[278] R ⁷ and R ¹² are both methyl;
[279] Ar is 2-methyl-1,3-thiazolinyl, 2-methyl-1,3-oxazolinyl, 2-hydroxymethyl-1,3-thiazolinyl, or 2-hydroxymethyl-1,3 Oxazolinyl;
[280] W is NH or O assuming at least one of R ¹ , R ⁵ , R ⁶ and R ¹¹ is not hydrogen.
[281] In another aspect of the invention, there is provided a compound having the formula:
[282]
[283] Wherein R ⁴ is hydrogen or oxo;
[284] Both R ⁵ and R ⁶ form hydrogen or a carbon carbon double bond together;
[285] R ⁷ is hydrogen or methyl;
[286] R ⁹ is hydrogen or hydroxyl;
[287] Both R ⁸ and R ^{10 form} hydrogen or a carbon carbon double bond or an epoxide together;
[288] W is O or NH;
[289] X is O or S;
[290] R ¹⁴ is methyl or hydroxymethyl.
[291] In another aspect of the invention, there is provided a compound having the formula:
[292] Formula IV
[293]
[294] In the formula,
[295] R ⁴ is hydrogen or oxo;
[296] R ⁵ and R ⁷ are each independently hydrogen or methyl;
[297] R ⁶ is hydrogen;
[298] R ⁸ and R ¹⁰ both hydrogen or together form a carbon carbon double bond or an epoxide; or
[299] R ⁶ and R ⁸ together form a double bond;
[300] R ⁹ is hydrogen, hydroxy or oxo;
[301] W is O or NH;
[302] X is O or S;
[303] R ¹⁴ is methyl or hydroxymethyl.
[304] In another embodiment of the invention, compounds are provided having the formula:
[305]
[306]
[307] [206] The compounds of the present invention can be used in any of a variety of non-limiting ways, including as anti-cancer agents as cytotoxyc agents. Example assays for evaluating the degree of tubulin polymerization and cytotoxicity are provided in Example 12.
[308] The compound of the present invention can be made using several methods. In one aspect of the invention, these compounds are produced by recombinant host cells expressing epothilone PKS. In one embodiment, compounds having the formulas are prepared by altering AT specificity in one or more modules and / or by changing enzyme domains in one or more modules, wherein R ¹ to R ¹² are defined in compounds of Formula I As shown):
[309]
[310] Example 11 describes the formula types and specific compounds that can be used in this method.
[311] In another embodiment of the present invention, the oxazole counterparts of formula (V) can be made by altering the fermentation conditions of the host cell, which will usually make the compound of formula (V). The thiazole moiety of the compound of formula V is derived from a cysteine binding site in NRPS. Epothilones H ₁ and H ₂ , the oxazole counterparts of epothilones C and D, are made in small amounts by host cells and are believed to occur by often binding to serine instead of cysteine in epothilone NRPS.
[312] The method of the present invention takes advantage of the apparent competition between serine and cysteine for the NRPS binding site of epothilone PKS and utilizes fermentation conditions that bind to serine instead of cysteine in epothilone NRPS. It has been found that when host cells are grown in serine-enriched (eg, about 50-fold concentrations of the basal concentration) medium, most of the oxazole-containing compounds are produced instead of the commonly produced thiazole-containing compounds. Eventually, by growing recombinant host cells engineered to make specific compounds or certain epothilone compounds of Formula V in serine-enriched media, these same cells now produce better oxazole counterpart compounds represented by Formula VI. do:
[313]
[314] That is, the method of the present invention is a method for obtaining two compounds at once and in a simple and refined manner at the same time, a compound corresponding to formula (V) and a corresponding compound corresponding to formula (VI). Serine reinforcement methods for preparing oxazole-containing compounds are described in more detail in Example 13. This example demonstrates the concentration of epothilones D and C, which are generally produced by strain K111-40-1, in order to enhance the production of epothilones H ₂ and H ₁ , the oxazole counterparts of epothilones D and C, respectively. Describe the conditions used to reduce. Other recombinant constructs for producing another compound of formula V of the present invention can also be grown using conditions similar to those for preparing compounds of formula VI.
[315] In another aspect of the invention, compounds are produced using a method called chemobiosynthesis. The method utilizes epothilone PKS modified in such a way that PKS accepts and binds synthetic precursors at designated locations. The synthetic precursor is then processed by PKS in a conventional manner from that point on.
[316] [212] There are the type of change required in chemical synthesis is illustrated in Example 9, Example 9 is a main product generally epothilone A, B, KS2 knockout mikso Lactococcus glass tooth strains producing the C and D ( knockout) Describes the version structure. KS2 knockout refers to the inactivation of the KS domain of extender module 2 such that the resulting PKS cannot load and process the product of the previous module, loading domain and NRPS (referred to as extender module 1). Eventually, PKS-directed synthesis ceases in the ACP of extender module 2 and in the absence of synthetic precursors no epothilone production occurs by this strain. However, when supplying the synthetic precursor to the strain, it mimics the loading domain and the product of extender module 1 so that the ACP of extender module 2 binds to the precursor and the PKS processes the precursor from that point. For example, the following synthetic precursors to the knockout strain of Example 9
[317]
[318] Providing will produce Epothilones B and D (Epothilones A and B are also produced, but only in minor amounts), as described in detail in Example 10. See also FIG. 1. If the knockout strain of Example 9 is provided with, for example, the following synthetic precursors,
[319]
[320] Wherein R is hydrogen, hydroxy, halogen, amino, C _1-5 alkyl, C _1-5 hydroxyalkyl, C _1-5 alkoxy, and C _1-5 aminoalkyl, more preferably hydrogen or methyl )
[321] The following epothilone compounds and their 12,13-epoxide counterparts are produced, respectively.
[322]
[323] Thus, by changing synthetic precursors, a single KS2 knockout strain can be used to make a wide range of compounds. Indeed, the synthetic precursor of the formula
[324]
[325] It can be used to make a compound having the following formula by providing.
[326]
[327]
[328] Wherein Ar is aryl and R ⁷ is hydrogen or methyl,
[329] Examples of suitable Ar groups include, but are not limited to:
[330]
[331] Wherein R is hydrogen, hydroxy, halogen, amino, C _1-5 alkyl, C _1-5 hydroxyalkyl, C _1-5 alkoxy, and C _1-5 aminoalkyl. In a more preferred embodiment, R is hydrogen or methyl. Example 10 describes the synthesis of various precursors and their use in chemical biosynthesis and their corresponding 12,13-epoxide counterparts, respectively.
[332] [214] In another embodiment, loading domain knockout is used to make certain compounds of the invention. For example, the loading domain knockout of the starting material used in Example 9 can also be used to make compounds of Formulas VII and VIII by providing a synthetic precursor of the formula:
[333]
[334] [215] In another embodiment, the KS2 or loading domain knockout of other strains of the invention comprises compounds comprising the strains described in Example 11, including but not limited to compounds having an aryl moiety other than 2-methyl thiazole. Used to make For example, feeding the synthetic precursor of Formula (IX) to the KS2 knockout of a structure that primarily produces 9-oxo-epothilone D will produce a compound having the formula
[335]
[336] In the formula, Ar is aryl.
[337] [216] In another aspect of the invention, compounds made from host cells expressing epothilone PKS can be further modified using biological and / or synthetic methods. In one embodiment, Ar is
[338]
[339] Phosphorus compounds of formula I can be hydroxylated at C-21 carbons using microbial-derived hydroxylases. Protocols for performing this transformation are described, for example, in PCT Publication No. It is described in WO 00/39276, the entire contents of which are referred to herein, in particular Example 14.
[340] [217] In another embodiment, compounds of the invention having carbon-carbon double bonds at positions corresponding to C-12 and C-13 of epothilone AD are epoxidized using EpoK or another P450 epoxidase. Can be. The general method of using EpoK for epoxidation is described in PCT Publication No. It is described in Example 5 of WO 00/31247, the contents of which are referred to herein, in particular Example 15. The epoxidation reaction can also occur by contacting an epothilone compound containing a double bond at a position corresponding to a bond between carbon-12 and carbon-13 to a cell culture expressing functional EpoK. Such cells include Soranium cellulose, myxobacterium . In a particularly preferred embodiment, the Soranium cellulose is expressing EpoK but does not contain functional epothilone polyketide synthase (“PKS”). Such strains can be made by mutations that render the gene inoperable due to mutations in one or more of the epothilone PKS genes. Such mutants may occur naturally (which can be found by screening) or by mutagens such as chemicals, light irradiation or genetic engineering. A particularly effective strategy for making these strains using nonfunctional epothilone PKS is homologous recombination as described in PCT Publication WO 00/31247.
[341] [218] In another embodiment, the epoxidation reaction may occur using synthetic methods. For example, as shown in Scheme 2, the desoxy compounds of the present invention can be converted to their epoxy counterparts by reacting these desoxy compounds with dimethyldioxirane.
[342]
[343] Example 16 describes this synthesis in detail.
[344] In another embodiment, the macrolactone of the present invention may be converted to the macrolactam of the present invention. As shown in Scheme 3, the dexyl macrolactone of the present invention is converted to its oxy corresponding compound by epoxidation using the dimethyldioxirane described in Scheme 2.
[345]
[346] Oxy-macrolactone is treated with sodium azide and tetrakis (triphenylphosphine) palladium to ring open to form azido acid. The azide is then reduced with trimethylphosphine to make an aminocarboxylic acid.
[347] [220] The epoxy-compound of the present invention, wherein W is NH, can be prepared by macrolactamating aminocarboxylic acid.
[348]
[349] As shown in Scheme 4, amino carboxylic acid is treated with 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide and 1-hydroxybenzotriazole to form epoxy-macrolactam. Deoxy- macrolactam can be made by treating epoxy-macrolactam with tungsten hexachloride and butyllithium.
[350] The epoxy-compound of the present invention, wherein W is NR ¹³ , wherein R ¹³ is not hydrogen, can be prepared by treating amino carboxylic acid with aldehyde and sodium cyanoborohydride prior to macrolactamation.
[351]
[352] As shown in Scheme 5, amino carboxylic acid is treated with aldehyde, R ¹³ HO and sodium cyanoborohydride to form a substituted amino acid, which is then optionally deoxylated as described for macrolactamation and Scheme 4 ¹³ produces epoxy and dexyl macrolactam, but not hydrogen.
[353] The synthesis for making the macrolactam of the present invention is described in more detail in Examples 17-19. Example 17 signs the making of amino acids using 9-oxo-epothilone D as an exemplary starting material. Examples 18 and 19 describe methods of making epoxy and dexoxy macrolactam versions of 9-oxo-epothilone D, respectively. Examples 20 and 21 describe methods of making epoxy and deoxy substituted macrolactam versions of 9-oxo-epothilone D, respectively.
[354] [223] The composition of the present invention generally comprises a compound of the present invention and a pharmaceutically acceptable carrier. The compounds of the present invention may be in free form or, where appropriate, in the form of pharmaceutically acceptable derivatives such as precursors and salts and esters of the compounds of the present invention.
[355] The composition of the present invention may be in any suitable form, such as in solid, semisolid or liquid form. See Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th edition, Lippicott Williams & Wilkins (1991). Generally, such pharmaceutical preparations will contain, as active ingredient, one or more compounds of the present invention in admixture with organic or inorganic carriers or excipients suitable for external, enteric or parenteral administration. The active ingredient can be mixed with non-toxic, pharmaceutically acceptable carriers, for example for general tablets, pellets, capsules, suppositories, pesticides, solutions, emulsions, suppositories, and any other suitable form. Carriers that can be used include water, glucose, lactose, acacia gum, gelatin, mannitol, starch paste, urea and any suitable carrier for preparation in solid, semisolid or liquid form. Auxiliary sterilizers, thickeners and coloring agents and flavorings may also be used.
[356] [224] In one embodiment, a composition containing a compound of the present invention comprises Cremophor It does not contain. Cremophor (BASF Aktiengesellschaft) is a polyethoxylated castor oil which is widely used as a surfactant for preparing low-soluble drugs. However, Cremophor Because it can cause allergic reactions in patients, the composition is Cremophor It is preferred to contain minimal or no at all. Cremophor Compositions containing epothilones A or B without It is described in WO 99/39694, incorporated herein by reference.
[357] If applicable, the compounds of the present invention may be formulated as microcapsules and nanoparticles. Microcapsules and Nanoparticles in Medicine and Pharmacy by Max Donbrow, ed., CRC Press (1992) and US Patent Nos. 5, 510, 118; 5, 534, 270; And 5, 662, 883, the general protocols are described and referenced in the text. By increasing the ratio of surface area to volume, these compositions are made suitable for oral delivery of the active compound, otherwise they are not suitable for oral delivery.
[358] The compound of the present invention may be formulated using other methods previously used for low-soluble drugs. For example, the compounds of the present invention may form emulsions with vitamin E or PEGylated derivatives thereof, as described in WO 98/30205 and 00/71163, already referenced herein. In general, the compounds of the present invention are dissolved in aqueous solutions containing ethanol (preferably less than 1% w / v). Vitamin E or PEGylated-vitamin E is added. Ethanol is then removed to form a pre-emulsion that can be formulated for intravenous or oral administration. Another method is to encapsulate the compound of the invention in liposome capsules. Methods of forming liposomes as drug delivery vehicles are well known in the art. PCT Publication No. Related to Epothilone B Suitable protocols for WO01 / 10413 and Taxol, a relatively insoluble anticancer agent, are described in US Pat. 5,683,715; 5,415,869; And 5,424,073, all of which are incorporated herein by reference. Among the various lipids that can be used, particularly preferred lipids for making epothilone-encapsulated liposomes include phosphatidylcholine and polyethyleneglycol-derived distearyl phosphatidylethanolamine. Example 22 shows a protocol for making liposomes containing 9-oxo-epothilone D and the method can be readily applied to making liposomes containing other compounds of the invention.
[359] Another method involves the composition of the compounds of the invention into drugs using polymers such as biopolymers or biocompatible (synthetic or naturally occurring) polymers. Biocompatible polymers are divided into biodegradable and non-biodegradable. Biodegradable polymers are degraded in vivo as a function of chemical composition, manufacturing method, and implant structure. Examples of synthetic polymers include polyhydroxyhydrides, polylactic acid, polyhydroxy acids such as polyglycolic acid and copolymers thereof, polyester polyamide polyorthoesters and some polyphosphazenes. Examples of naturally occurring polymers include polysaccharides and proteins such as gelatin, albumin, hyaluronic acid, collagen.
[360] Another method is to conjugate the compounds of the present invention to polymers which increase water solubility. Examples of suitable polymers include polyethylene glycol, poly- (d-glutamic acid), poly- (l-glutamic acid), poly- (l-glutamic acid), poly- (d-aspartic acid), poly- (l-aspartic acid) , Poly- (l-aspartic acid), and copolymers thereof. Polyglutamic acid having a molecular weight of about 5,000 to about 100,000 is preferred, more preferably about 20,000 to about 80,000, and most preferred is polyglutamic acid having a molecular weight of about 30,000 to 60,000. The polymer is conjugated via an ether bond to the hydroxyl of one director of the epothilones of the present invention using the protocol described essentially in US Pat. No. 5,977,163 (see text, in particular Example 23). Preferred conjugation sites include, for the 21-hydroxyl derivatives of the invention, hydroxyl off carbon-21. Other conjugation sites include hydroxyl off carbon 3 and hydroxyl off carbon 7.
[361] [229] In another method, the compound of the present invention is conjugated to a monoclonal antibody. By this method, the compounds of the present invention can be targeted to specific targets. General protocols for the design and use of conjugated antibodies are described in Michael L. Grossbard, Monoclonal Antibody-Based Therapy of Cancer (1998) and referenced herein.
[362] The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the patient to be treated and the particular mode of administration. For example, a composition for intravenous administration may contain about 1 mg / mL to about 25 mg / mL, preferably about 5 mg / mL to l 15 mg / mL, more preferably about 10 mg / mL of a compound of the present invention. It is contained in an amount. Intravenous compositions are usually diluted in normal saline or 5% dextrose solution at a rate of about 2 to about 30 times prior to use.
[363] [231] In one aspect of the invention, the compounds of the invention are used to treat cancer. In one embodiment, the compounds of the present invention are used to treat cancers of the head and neck, such as tumors of the head, neck, nasal cavity, sinus, nasopharynx, oral cavity, middle head, larynx, hypopharyngeal, salivary glands, and paraganglioma. Is used. In another embodiment, the compounds of the present invention are used to treat liver and biliary tree, in particular hepatocellular carcinoma. In another embodiment, the compounds of the present invention are used to treat bowel cancer, particularly rectal cancer. In another embodiment, the compounds of the present invention are used to treat ovarian cancer. In another embodiment, the compounds of the present invention are used to treat small cell and non-small cell lung cancer. In another embodiment, the compounds of the present invention are used to treat breast cancer. In another embodiment, the compounds of the present invention are used to treat fibrosarcomas, larval fibrous histiocytoma, fetal rhabdomyosarcoma, leiomyosarcoma, neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma and acinar soft sarcoma. In another embodiment, the compounds of the present invention are used to treat neoplastic tissue of the central nervous system, in particular brain cancer. In another embodiment, the compounds of the present invention comprise Hodgkin 'lymphoma, lymphoblastic lymphoma, cystic lymphoma, mucous-associated lymphoid tissue lymphoma, mantle cell lymphoma, B-Lineage large cell lymphoma, Burkitt lymphoma, and T- Cell Undifferentiation Used to treat lymphomas, including large cell lymphomas.
[364] [232] This method consists in administering a therapeutically effective amount of a compound of the invention to a subject with cancer. This method can be repeated as needed to suppress cancer (ie prevent further growth) or to remove the cancer. Clinically, the practice of the present invention will reduce the size and number associated with cancer growth and / or related symptoms (if applicable). Pathologically, the practice of the present invention will result in at least one of the following: inhibiting cancer cell proliferation, reducing cancer or tumor size, preventing further metastasis, and inhibiting neovascularization of the tumor.
[365] The compounds and compositions of the present invention can be used in combination therapy. That is, the compounds and compositions of the present invention may be administered simultaneously, prior to, or subsequently to one or more other desired treatments or medical treatments. Certain combinations of therapies and modalities in a combination therapy should be made in view of the possibility of coexistence of therapies and / or modalities and the desired therapeutic effect to be achieved.
[366] [234] In one embodiment, the compounds and compositions of the present invention are used in combination with other anticancer agents or therapies. Non-limiting examples of other anticancer agents include: (i) mechloretamine, chlorambucil, cyclophosphamide Alkylating drugs such as melphalan, ifosfamide; (ii) antimetabolic agents, such as methotrexate; (iii) vinblastine, paclitaxel, desotaxel, and discodemolide and fine microtubule stabilizers; (iv) angiogenesis inhibitors; And (v) cytotoxic antibiotics such as doxorubicon (Adriamycin), bleomycin and mitomycin. Examples of other chemotherapy include: (i) surgery; (ii) radiotherapy; And (iii) photodynamic therapy.
[367] In another embodiment, the compounds and compositions of the present invention are used in combination with a medicament or method for alleviating potential side effects that may result from the compounds or compositions of the present invention, such as diarrhea, nausea and vomiting. . Diarrhea can be ruled with antidiarrheal agents such as opioid preparations (such as codeine, diphenoxylate, diphenoxine and loeramide), bismuth subsalicylate and octreotide. Nausea and vomiting can be treated with anti-emetic agents such as dexamethasone, metoclopramide, diphenylhydramine, lorazepam, ondansetron, prochlorperazine, thiethylperazine and dronabinol. Cremophor For compositions comprising polyethoxylated castor oils, such as anaphylaxis using an H ₁ antagonist and / or H ₂ antagonist such as corticosteroids and / or diphenylhydramine HCl, such as dexamethasone and methylprednisolone Could be mitigated. Intravenous administration uses and descriptions of pretreatment regimes are presented in Examples 24 and 25, respectively.
[368] [236] In another aspect of the invention, the compounds of the invention are used to treat a disease other than cancer characterized by cell hyperproliferation. In one embodiment, the compounds of the present invention are used to treat psoriasis, ie, psoriasis characterized by cellular hyperproliferation of keratinocytes forming a swelling lesion on the skin. The methods of the present invention comprise administering to a patient suffering from psoriasis a therapeutically effective amount of a compound of the present invention. This method can be repeated as needed to reduce the number or severity of the lesion or until the lesion is removed. Clinically, this method can be used to reduce the size or number of skin lesions, reduce skin symptoms (pain, burns, and bleeding of the skin to be treated) and / or related symptoms (eg, joint redness, fever, swelling, diarrhea). , Abdominal pain) will be achieved. Pathologically, by practicing the method of the present invention, at least one of the following results will be obtained: inhibition of keratinocyte proliferation, reduction of skin inflammation (eg, growth factor, antigen presentation, reactive oxygen spicesis production and matrix metalloproteinases). By affecting the agent), and inhibition of cutaneous angiogenesis.
[369] [237] In another embodiment, the compounds of the present invention are used to treat multiple sclerosis, a symptom characterized by demyelination progression in the brain. Although the exact mechanisms involved in the loss of myelin are not understood, an increase in astrocyte proliferation and accumulation at the site of myelin destruction occurs. At this site, there is increased macrophage-like activity and at least partly protease activity responsible for the degradation of myelin myelin. The method of the invention consists in administering a therapeutically effective amount of a compound of the invention to a subject suffering from multiple sclerosis. The method may be repeated as necessary to inhibit astrocytic proliferation and / or reduce the severity of motor function and / or prevent or attenuate chronic progression of the disease. Clinically, the practice of the present invention can lead to visual symptoms (visual impairment, diplopia), gait-related disorders (weakness, axial instability, sensory impairment, convulsions, paralysis, dexterity loss), upper extremity disorders (weakness, Symptoms of convulsions, sensory impairment), bladder disorders (emergency, urinary incontinence, hesitation, incomplete urination), depression, psychological anxiety, and cognitive impairment will be improved. Pathologically, the method of the present invention results in the loss of myelin, the destruction of the extra-blood barrier, perivascular infiltration of mononuclear cells, immunodeficiency, gelatinous wound formation and astrocytic proliferation, metalloproteinase production, and impaired conduction rate. One or more of the symptoms will be alleviated.
[370] In another embodiment, the compounds of the present invention are used to treat inflammatory diseases that lead to rheumatoid arthritis treatment, chronic multisystem, regression, and sometimes to joint stiffness and destruction of joints in need of treatment. Rheumatoid arthritis is characterized by marked thickening of the synovial membrane, which forms ciliary processes that extend into the joint space, lamination of the synovial lining (metaplastic cell proliferation), synovial infiltration by leukocytes (macrophages, lymphocytes, plasma cells and lymphoid cysts; "inflammatory Synovialitis), and fibrin deposition accompanied by intracellular syndrome necrosis. The tissue formed as a result of this process is called pannus, which actually grows until it fills the joint space. Panus causes an excessive network of new blood vessels through angiogenesis processes that are essential for the onset of synovitis. Other mediators of inflammatory processes from panus tissue cells (eg, hydrogen peroxide, superoxide, lysosomal enzymes, and arachidonic acid metabolites) and release of digestive enzymes (matrix metalloproteinases (eg collagenase, stromellisin) )) Gradually destroys cartilage tissue. Panus penetrates into the articular cartilage, causing corrosion and fragmentation of the cartilage tissue. Indeed, erosion of the subchondral bone occurs, which may be fibrous arthroplasty of the associated joint and ultimately arthroscopic stiffness of the bone.
[371] [239] The method of the present invention is to administer a therapeutically effective amount of a compound of the present invention to a patient suffering from rheumatoid arthritis. This method may be repeated as necessary until inhibition of synovial cell proliferation and / or reduction in the severity of loss of motility of the joint to be treated and / or prevention or alleviation of chronic progression of the disease. Clinically, the method of the present invention may be practiced to achieve one or more of the following: (i) Severity of symptoms (pain, swelling and tenderness of related joints; morning stiffness, weakness, fatigue, loss of appetite, weight loss) Reduction of; (ii) reduction of clinical signs of this disease (thickening of joint capsules, synovial hypertrophy, joint exudates, soft tissue buildup, reduced activity radius, arthroscopy and arterial immobilization); (iii) reduction of extra-articular manifestations of this disease (rheumatic nodule, vasculitis, pulmonary nodule, intervening fibrosis, pericarditis, episcleitis, iris, Felty syndrome, osteoporosis); (iv) an increase in the frequency and duration / asymptomatic period in which the disease presents; (v) prevention of immobilization of disorders and disorders; and / or (vi) prevention / mitigation of chronic progression of this disease. Pathologically, the method of the present invention may yield at least one of the following results: (i) reducing the inflammatory response; (ii) disrupting inflammatory cytokine (IL-1, TNFa, FGF, VEGF) activity; (iii) inhibits synovial cell proliferation; (iv) inhibit matrix metalloproteinase activity, and / or (v) inhibit angiogenesis.
[372] [240] In another embodiment, the compounds of the present invention are used to treat atherosclerosis and / or restenosis, particularly in patients who can treat occlusion with an intravascular stent. Atherosclerosis is a chronic vascular injury that normally causes "cancer-like" changes in the characteristics of some of the normal vascular smooth muscle cells ("VSMCs") in the artery walls that control blood vessel tone control blood flow. These VSMCs proliferate abnormally, secreting substances (growth factors, tissue-degrading enzymes, and other proteins) that can infiltrate and diffuse into the lining of the blood vessels, blocking blood flow, thereby causing local blood clots Completely abnormally occludes the restenosis, that is, restenosis, which is caused by stenosis or arterial stenosis after corrective treatment.
[373] This method consists of coating a stent with a therapeutically effective amount of a compound of the invention to deliver the stent to a diseased artery of a subject suffering from atherosclerosis. Methods of coating compounds on stents are described, for example, in US Pat. 6,156,373, and 6,120,847. Clinically, at least one of the following will be achieved by practicing the method of the present invention: (i) increased blood flow in the artery; (ii) reducing the severity of clinical symptoms of the disease; (iii) reduced restenosis rate; Or (iv) prevention / mitigation of chronic progression of atherosclerosis. Pathologically, the practice of the present invention will achieve at least one of the following at the stent graft site: (i) decrease the inflammatory response, (ii) inhibit VSMC secretion of matrix metalloproteinases; (iii) inhibiting smooth muscle cell accumulation; And (iv) VSMC phenotype undifferentiation.
[374] [242] In one embodiment, the dosage administered to a patient suffering from a disease other than cancer characterized by cancer or cell proliferation can be from about 1 mg / m ² to about 200 mg / m ² , which is Every week, every two weeks, every three weeks, as a bolus (with any appropriate route of administration) or continuous infusion (eg, 1 hour, 3 hours, 6 hours, 24 hours, 48 hours or 72 hours). May be administered. However, it will be appreciated that the specific dosage for a particular patient may vary depending on several factors. Such factors include the activity of the specific compound employed; The age, body weight, general health, sex, and dietary level of the subject; Drug excretion and route of administration; Whether it is used in combination with other drugs for treatment; And the severity of the condition being treated.
[375] [243] In another embodiment, the dosage is about 10 mg / m ² to about 150 mg / m ² , preferably about 10 mg / m ² to about 75 once every three weeks, as needed and tolerated mg / m ² , more preferably about 15 mg / m ² to about 50 mg / m ² . In another embodiment, the dosage is about 1 mg / m ² to about 150 mg / m ² , preferably about 10 mg / m ² to about 75 mg / m once every two weeks as needed and tolerated ² , more preferably about 25 mg / m ² to about 50 mg / m ² . In another embodiment, the dosage is about 1 mg / m ² to about 100 mg / m ² , preferably about 5 mg / m ² to about 50 mg / m once a week as needed and tolerated ² , more preferably about 10 mg / m ² to about 25 mg / m ² . In another embodiment, the dosage is about 0.1 mg / m ² to about 25 mg / m ² , preferably about 0.5 mg / m ² to about 15 mg / m ² , once a day as needed and tolerated, More preferably about 1 mg / m ² to about 10 mg / m ² .
[376] The above detailed description and the following embodiments are provided for purposes of explanation of the present invention, but the scope or claims of the present invention are not limited thereto.
[390] Example 1
[391] Myxococcus xanthus) Preparation of Expression Vector
[392] [245] DNA providing the insertion and attachment function of phage Mx8 was inserted into commercially available pACYC184 (New England Biolabs). Feb. of Salmi et al. 1998, J. Bact. ˜2360 bp Mfe I- Sma I was isolated from plasmid pPLH343 and conjugated to the large Eco RI- Xmn I restriction enzyme fragment of plasmid pACYC184 as described in 180 (3): 614-621. The circular DNA thus obtained was ˜6 kb in size and was named Plaskeid pKOS35-77.
[393] Plasmid pKOS35-77 serves as a convenient plasmid for expressing the recombinant PKS gene of the invention under the control of the epothilone PKS gene promoter. In one embodiment, the entire epothilone PKS gene with homologous promoter is inserted into one or more fragments of this plasmid to produce the expression vector of the present invention.
[394] [247] The present invention also provides expression vectors in which the recombinant PKS gene of the present invention is regulated by the Myxococcus xanthus promoter. For the production of an exemplary vector, it did remove the promoter of the pilA gene of Lactococcus mikso glass tooth as a PCR amplification product. pilA gene, including the promoters of Wu and Kaiser, Dec. 1997, J. Bact. Plasmid pSWU357 as described in 179 (24): 7748-7758 was mixed with PCR primers Seq1 and Mxpil1 primers:
[395] Seql: 5'-AGCGGATAACAATTTCACACAGGAAACAGC-3 '(SEQ ID NO: 1) and Mxpill: 5'-TTAATTAAGAGAAGGTTGCAACGGGGGGC-3' (SEQ ID NO: 2),
[396] Amplification using standard PCR conditions yielded ˜800 bp fragments. This fragment was cut with the restriction enzyme Kpn I, and joined to the large KpnI-EcoRV restriction fragment of commercially available plasmid pLitmus 28 (New England Biolabs) is. The resulting circular DNA was named plasmid pKOS35-71B.
[397] [248] The promoter of the pilA gene from plasmid pKOS35-71B was isolated as a ~ 800 bp Eco RV- Sna BI restriction enzyme fragment and conjugated to the large MSc I restriction enzyme fragment of plasmid pKOS35-77 to circumscribe 6.8 kB of circular DNA. Got it. Since the ˜800 bp fragment can be inserted in either direction, this conjugation yielded two plasmids of the same size, which were named plasmids pKOS35-82.1 and pKOS35-82.2. Restriction site and function map of these plasmids are shown in FIG.
[398] [249] Plasmids pKOS35-82.1 and pKOS35-82.2 serve as convenient starting materials for the vectors of the present invention in which recombinant PKS genes are placed under the control of the Myxococcus xanthus pilA gene promoter. These plasmids contain a single PacI restriction enzyme recognition site located immediately downstream (downstream) of the transcription initiation site of the promoter. In one embodiment, the entire epothilone PKS gene without homologous promoters is inserted into one or more fragments into these plasmids at PacI restriction enzyme sites to obtain expression vectors of the invention.
[399] [250] The pilA promoter sequence in these plasmids is as follows:
[400]
[401] [251] To make a recombinant mikso Lactococcus glass tooth host of the present invention cells in 300 rpm at 30 ℃ until the Klett 100 CYE the mikso Lactococcus glass tooth media (Campos and Zusman, 1975, Regulation of development in Myxococcus xanthus: effect of 3 ': 5'-cyclic AMP, ADP, and nutrition, Proc. Natl. Acad. Sci. USA 72: 518-522. The rest of the protocol is carried out at 25 ° C. unless stated otherwise. Cells are pelleted by centrifugation (10 min at 8000 rpm in SS34 or SA600 rotor) and resuspended in deionized water. The cells are pelleted again and resuspended to 1/100 of the original volume.
[402] DNA (1 to 2 μL) is electrophoresed into cells in 0.1 cm cuvettes at room temperature, 400 ohms, 25 μFD, 0.65 V over a period of time ranging from 8.8 to 9.4. The DNA is salt free and distilled to resuspend in deionized water or dialyzed on a 0.025 μm Type VS membrane (Millipore). For low efficiency electrophoresis, DNA is spot-dialysis and overgrows in CYE. Immediately after electrophoresis, 1 mL of CYE is added and 1.5 mL of CYE, once added to the cells in the cuvette (2.5 ml total volume), is placed in a 50 mL Erlenmeyer flask. Cells were grown at 30-32 ° C., 300 rpm for 4-8 hours (or overnight) to allow for the selection of selectable markers. The ferro are then plated and sorted on CYE soft agar. When using a selectable marker Los kanamycin, it is the most common production was 10 ³ to 10 ^5/1 μg DNA. When streptomycin is used as a selection marker, it is included in the agar top because it binds to agar.
[403] [253] By this method, the recombinant DNA expression vector of the present invention was transferred into a myxococcal host cell expressing the recombinant PKS of the present invention and producing an epothilone, an epothilone derivative and other novel polyketides encoded by the present invention. Move it.
[404] Example 2
[405] Bacterial Artificial Chromosome (BAC) and Chromosome Insertion for Epothilone Expression in Myxococcus xanthus
[406] [254] Myxococcus goblet , which is closely related to Sorangium cellulosum and can use several cloning vectors, for the expression of epothilone PKS and a modified enzyme gene in a heterologous host to produce epothilone by fermentation. Tooth (Myxococcus xanthus ) is used in the method of the present invention. Since Myxoscus xanthus and Soranium cellulose are both myxobacteria, they can share common elements in gene expression, translation control, and post-translational modifications. Myxococcus xanthus was developed for gene cloning and expression: DNA is introduced by electrophoresis and several vectors and genetic markers are available for the introduction of foreign DNA, including those allowing their stable insertion into chromosomes. Myxococcus xanthus can be grown relatively easily in a complex medium of fermenter and, if necessary, can be manipulated to increase gene expression.
[407] [255] Myxococcus xanthus can assemble a complete gene cluster by binding the epothilone cluster to the chromosome by homologous recombination to introduce the epothilone gene clotor into it. On the other hand, a complete cluster of epothilone genes can be cloned onto the bacterial artificial chromosome (BAC) and then inserted into Myxococcus xanthus for insertion into the chromosome.
[408] [256] cosmid small band, from these cosmids to create a gene cluster from pKOS35-70.1A2 and pKOS35-79.85 (~ 2kb or greater) it was introduced into the cup mikso Caucus tooth for a gene cluster of larger fragments Create a recombination site. As shown in Figure 3, plasmids pKOS35-154 and pKOS90-22 are made to introduce these recombinant sites. The strategy for assembling the epothilone gene cluster in the chromosome of Myxococcus xanthus is shown in FIG. 4. First, select the neutral site of the bacterium that does not destroy any genes or transcription units. One such band is a downstream gene of devS, the bar does not appear to affect the growth and development of mikso Caucus tooth mug. First to linearize the plasmid pKOS35-154 into Dra I then electrophoresed into mikso Lactococcus glass tooth. This plasmid contains two zones of the dev position flanking two fragments of the epothilone gene cluster. A cassette consisting of the kanamycin resistance marker and the E. coli galK gene is inserted between the epo gene bands. See Ueki et al., 1996, Gene 183: 153-157. Kanamycin resistance occurs in colonies when DNA recombines into the dev band by double recombination using the dev sequence as a homology band.
[409] This strain, K35-159, contains a small (~ 2.5 kb) band of clusters of epothilone genes that allow recombination of pKOS35-79.85. Since the resistance markers on pKOS35-79.85 are the same as the resistance markers on K35-159, tetracycline transposons were transposed into cosmids and cosmids containing this transposon inserted into kanamycin markers were selected. This cosmid pKOS90-23 was electrophoresed into K35-159 and oxytetracycline resistant colonies were selected to make strain K35-174. Cells were plated on CYE plates containing 1% galactose to remove unwanted zones from cosmid and leave only the epothilone gene. In the presence of the galK gene, cells are sensitive to 1% galactose. Galactose resistant colonies of K35-174 lost the galK marker by mutation or recombination in the galK gene. When recombination occurs, galactose resistant strains become sensitive to kanamycin and oxytetracycline. Strains sensitive to both antibiotics are identified by Southern blot analysis. The correct strains were identified and named K35-175, which contain clusters of eponylone genes from 7 to 4680 bp downstream of the stop codon of epoK .
[410] In order to introduce modules 1 to 7, the process is repeated once more. Plasmid pKOS90-22 was digested with DraI and electrophoresed into K35-175 to make K111-13.2. This strain is electrophoresed with tetracycline resistant versions of pKOS35-70.1A2, pKOS90-38 to select colonies resistant to oxytetracycline. In this way, strain K111-13.23 is produced. Recombinants with the entire epothilone gene cluster are now screened using resistance to 1% galactose. This results in clones K111-32.25, K111-32.26, and K111-32.35. Strain K111-32.25 was prepared according to the Budapest Treaty under American Type Culture Collection, 10801 University Blvd. Manassas, VA, 20110-2209 USA, dated April 14, 2000, was assigned accession number PTA-1700. This strain contains both the epothilone gene and its promoter (s).
[411] [259] The fermentation was carried out by inoculating the strain into 5 mL of CYE (10 g Catonone per liter, 5 g yeast extract, and 1 g MgSO ₄ · 7H ₂ O) in a 50 mL flask to allow the culture to grow to the middle of the log phase. . 100 μL aliquots were dispensed into the CTS plates and the plates were incubated at 32 ° C. for 4-5 days. To extract the epothilones, agar and cells were impregnated from the plate and placed in a 50 mL conical test tube and acetone was added until the test tube was filled. The solution was incubated with rocking for 4-5 hours, the acetone was evaporated and the remaining liquid was extracted twice with the same volume of ethyl acetate. Water was removed from the ethyl acetate extract by adding magnesium sulfate. Magnesium sulphate was filtered off and evaporated until the liquid was dry. Epothilones were resuspended in 200 μL of acetonitrile and analyzed by LC / MS. Analysis showed that the strain produced epothilones A and B. Epothilone B was produced in an amount of about 0.1 mg / L in culture and Epothilone A was at a concentration of 5-10 times lower than this.
[412] This strain can also be used to produce epothilones in liquid media. Flasks containing CYE were inoculated with the epothilone producing strain. The following day, while the cells were in the middle of the log phase, the 5% inoculum was mixed with 1 mg / mL serine, alanine, and glycine and 0.1% sodium pyruvate at 0.5% CMM (0.5% Kaziton, 0.2% MgSO ₄ 7H ₂ 0, 10 mM MOPS pH 7.6) is added to the flask containing. Sodium pyruvate can be added up to 0.5% to increase the productivity of epothilone B, but this leads to a decrease in the ratio of epothilone B to epothilone A. Cultures are grown at 30 ° C. for 60-72 hours. The longer the incubation, the less epothilone titers. To volatilize the epothilones, the cultures are centrifuged at 10,000 rpm for 10 minutes in the SS34 rotor. The supernatant is extracted twice with ethyl acetate and rotary evaporated (“rotavaped”) until dry. This liquid culture produced 2-3 mg / L of nonmelting furnace similar to that observed for plate culture of epothilones A and B. When XAD (0.5-2%) was added to the culture, epothilones C and D were observed, epothilone D was observed to be present at 0.1 mg / L and epothilone C at 5-10 times lower concentrations. .
[413] In order to clone the entire gene cluster as one fragment, an artificial chromosome (BAC) library of bacteria is prepared. First, SMP44 cells are inoculated with agarose and lysed according to the BIO-RAD genomic DNA plug kit. DNA plugs are partially cut with restriction enzymes such as Sau3AI or HindIII and electrophoresed on FIGE or CHEF gels. DNA fragments are isolated by electroeluting the DNA fragments from agarose or by using gelase to degrade agarose. Selection methods for isolating fragments are described in Strong et al., 1997, Nucleic Acids Res. 19 : 3959-3961 (referenced herein) to electroelute. DNA is conjugated into BAC (pBeloBACII) digested with appropriate enzymes. A map of pBeloBACII is shown in FIG. 5.
[414] [262] DNA Sheng et al., 1995, Nucleic Acids Res . 23: Electrophoresis of DNA into DH10B cells following the method of 1990-1996 (referenced herein) resulted in the generation of Solanzium cellulose island genome libraries. Colonies are screened using probes from the NRPS band of the epothilone cluster. Positive clones were selected to isolate DNA for restriction enzyme analysis to confirm the presence of complete gene clusters. This positive clone was named pKOS35-178.
[415] [263] The plasmids pKOS90-22 and pKOS35-154 contain bands homologous with the upstream and downstream of the epothilone gene cluster cranked by the dev position to make strains usable for introducing pKOS35-178. Similarly, plasmid pKOS35-164 containing kanamycin resistant galK cassette was made. Kafeshi et al., 1995, Mol. Microbiol. 15: according to the method of 483-494, by cutting the plasmid with Dra I made K35-183 by electrophoresis into mikso Lactococcus glass tooth. This plasmid pKOS35-178 can be introduced into K35-183 by transduction with bacteriophage P1, or by electrophoresis and selected chloramphenicol resistant colonies. On the other hand, a version of pKOS35-178 containing the origin of a fusion transfer from pRP4 was made to deliver DNA from E. coli to K35-183. This plasmid is prepared by first making a transposon containing an oriT band from RP4 and a tetracycline resistance marker from pACYC184 and then transposing the transposon into pKOS35-178 in vitro or in vivo. This plasmid was transformed into S17-1 and conjugated to Myxococcus xanthus . This strain K35-190 was grown in the presence of 1% galactose to screen whether a second recombination occurred. This strain contains not only the potential promoter but also the entire epothilone gene. This strain was fermented and tested for the productivity of epothilones A and B.
[416] On the other hand, the transposon can be recombined into BAC by transposing the transposon into a temperature sensitive plasmid pMAK705 or pKO3 to select tetR and camS plasmids; Recombination is described in Hamilton et al., Sep. 1989, J. Bact. 171 (9): 4617-4622 and Link et al., Oct. 1997, J. Bact. 179 (20): 6228-6237 (all referenced in the text).
[417] In addition to inserting the pKOS35-178 into the dev position, it can also be inserted into the phage attachment site using the insert function from myxophage Mx8 or Mx9. and transposons were prepared containing the att site and insertion gene from MX8 or Mx9 with tetracycline gene from pACYC184. Another version of this transposon may have only an attachment site. In this version, the transgene is transfected by electrophoresis of the plasmids containing the transgene together or by transduction of the transfected protein from any constitutive promoter, such as the mgl promoter, in an electrophoretic strain (Magrini et al., Jul. 1999, J. Bact. 181 (13): 4062-4070, referenced herein). Once the transposon is prepared, it is transposed into pKOS35-178 to make pKOS35-191. This plasmid is introduced into Myxococcus xanthus as described above. This strain contains not only the latent promoter but also the entire epothilone gene. This strain was fermented to test the production of epothilones A and B. On the other hand, a strain containing the att site and the oriT band transposed to a BAC and can be conjugated to the resulting BAC into mikso Lactococcus glass tooth.
[418] [266] Once the epothilone gene is established in Myxococcus xanthus strains, manipulation of any part of the gene cluster, such as promoter alteration or module change, can be performed using kanamycin resistance and galK cassettes, as described below. . Myxococcus xanthus cultures containing the epo gene were grown in several media to test for epothilone productivity. If the yield of epothilones (particularly B or D) is low, myxococcus xanthus -producing clone medium was developed and mutant based strains were developed, as described in the following examples.
[419] Example 3
[420] Production and Purification Methods of Epothilones
[421] A. Myxocaucus XanthusHeterogeneous Productivity Optimization of Epothilone D in
[422] [267] The heterogeneous productivity of epothilone D in Myxococcus xanthus was increased by 140 times from titer of 0.16 mg / L by performing incorporation of adsorbent resin, identification of appropriate carbon source, and injection-batch process. .
[423] To reduce degradation of epothilone D in basal medium, XAD-16 (20 g / L) was added to stabilize extracellular product. This made it much easier to recover and increased the yield by three times. The use of oil as a carbon source for cell growth and product formation was also evaluated. Screening of various oils revealed that methyl oleate had the greatest effect. At 7 mL / L, the optimal concentration in the batch process, the maximum cell density increased from 0.4 g dry cell weight (DCW) / L to 2 g DCW / L. Yield depends on the presence of trace elements in the production medium. The external supply of trace elements to the basal medium resulted in an eight-fold increase in the peak titer of epothilone D, demonstrating the role of metal ions in cell metabolism and epothilone biosynthesis. To further increase production yield, a continuous ped-batch process was used to keep the cell density higher and the production period longer. Cell density was maintained at 7 g DCW / L by optimized fed-batch culture and the mean production titer was 23 mg / L.
[424] [269] epothilone is dynamic simple Te Solarium turmoil the secondary metabolites that are naturally produced by various strains of erase cellulite Rossum (Gerth et al., 1996; et al Gerth, 2001; references cited in example reference is the Listed at the end of the section and referenced in the text). These are potent inhibitors of microtubule depolymerization and their activity mechanism is similar to that of the anticancer drug Taxol (Bollag et al., 1995). Their cytotoxic effects on multi-drug resistant tumor cell lines expressing P-glycoprotein have made them potential therapeutic compounds of enormous commercial value (Su et al., 1997; Kowalski et al., 1997). They also have relatively high solubility, making it easy to formulate them for clinical evaluation.
[425] [270] Epothilones A and B are major fermentations of natural hosts (Gerth et al., 1996). Macrocycle cores of this polyketide molecule are formed by successive decarboxylation condensation of acetate and propionate units (Gerth et al., 2000). Epothilones A and B differ from one methyl group at the C-12 position of their carbon skeleton. This structural difference is the result of the insertion of acetate in the structure of Epothilone A and propionate in Epothilone B. Epothilones C and D are intermediates obtained during the biosynthetic pathway of epothilones A and B, respectively (Tang et al., 2000; Molnar et al., 2000). They are secreted as small amounts of produced radish during fermentation, totaling about 0.4 mg / L. Since in vivo studies have shown that epothilone D is the most promising antitumor among the four compounds above (Chou et al., 1998), large-scale production of this molecule is of great interest.
[426] [271] By sequencing the gene cluster responsible for the biosynthesis of epothilones (Tang et al., 2000; Molnar et al., 2000), Myxococcus xantu is a microbial host that is more closely related to Soranium cellulose and more suitable for genetic manipulation. Was used to produce these compounds. To promote epothilone D production, a deletion mutant strain (described below in Example 4) of this recombinant strain was prepared to catalyze the conversion of P450 epoxidase to catalyze the conversion of epothilones C and D into epothilones A and B, respectively. Inactivated (Tang et al., 2000). This genetic manipulation effectively promoted the secretion of epothilone C-D as the only fermentation product of Myxococcus xanthus , where the ratio of epothilone D to C was 4: 1. The resulting mutants were much more advantageous than natural hosts in both the recovery and purification of the desired product. This example describes a medium composition and fermentation strategy that increase the productivity of epothilone D 140 times in Myxococcus xanthus .
[427] [272] Adsorbent resins were used for myxobacteria fermentation to continuously capture small quantities of biologically active molecules (Reichenbach and Hoefle, 1993). To facilitate separation of epothilone D, hydrophobic resin XAD-16 was added to the culture medium. The recovery of the product has been greatly simplified since the bound product is easily eluted from the resin when the appropriate solvent is used. In addition, the use of XAD-16 minimizes epothilone degradation through product stabilization.
[428] [273] Myxococcus xanthus consists mainly of enzymatic hydrolysates of casein, such as peptone and kaziton , and has been traditionally cultured in a medium dependent on amino acids as the only carbon and nitrogen source (Reichenbach and Dworkin, 1991). As a result, ammonia accumulates in the fermentation broth due to the decomposition of amino acids. Gerth et al. (1986) found that extracellular ammonia concentrations of 35-42 mM in Myxococcus virescens cultures surprisingly corresponded to intracellular ammonia concentrations of 80-140 mM. More importantly, continuous removal of excess ammonia below 8 mM using an in situ membrane process has shown a dramatic increase in cell mass and secondary metabolite production (Hecht et al., 1990). Since high concentration production of ammonia is believed to inhibit the growth of myxococcus xanthus and epothilone D production, alternative carbon sources are desired to reduce amino acid consumption.
[429] [274] Although an adaptation process is required, extensive screening of oils has identified methyl oleate as a substrate that can be metabolized by Myxococcus xanthus . The addition of an external trace element solution to the growth medium increased the production of epothilone D by eight-fold in simple batch fermentation, yielding 3.3 mg / L. To further optimize this process, a fed-batch approach using intermittent or continuous infusion of cajiton and methyloleate was adopted to extend the production phase of the cells. The results obtained using two different injection strategies were compared with one another in this example.
[430] Materials and methods
[431] Inoculum Preparation
[432] [275] In order to produce epothilone D in a culture medium containing no methyl oleate, 20% (v / v) glycerol in mikso Lactococcus glass tooth strains the cells frozen 1 mL, 50-mL glass culture of K111-40.1 Contact with 3 mL of CYE medium at pH 7.6 consisting of 10 g / L Cagiton (Difco), 5 g / L yeast extract (Difco), 1 g / L MgSO ₄ .7H ₂ O and 50 mM HEPES in vitro. Cells were incubated for 3 days using a rotary shaker at 175 rpm at 30 ° C. This was transferred to a 250-mL Erlenmeyer flask containing 50 mL of CYE and incubated for 2 days under the same conditions. The seed culture was used to inoculate the 50-mL production flask at 5% (v / v) inoculum.
[433] In order to incubate myxococcus xanthus in a medium containing methyl oleate, cells had to be adapted to grow in the presence of oil. 3 mL of CYE medium supplemented with 3 μL of methyl oleate (Emerest 2301) (Cognis Corp.) was inoculated with 1 seed vial of frozen cells. Cells were grown in glass culture test tubes for 2-6 days at 30 ° C., 175 rpm, until the cultures were sufficiently dense when viewed under a microscope. Subsequently, CYE-MOM medium 50 at pH 7.6 consisting of 10 g / L Cagiton (Difco), 5 g / L yeast extract (Difco), 1 g / L MgSO ₄ .7H ₂ O, 2 mL / L methyl oleate, and 50 mM HEPES. The cells were transferred to 250 mL Erlenmeyer flasks containing mL. After 2 days of culture, cells were cryopreserved at −80 ° C. as 1 mL aliquots in 20% (v / v) glycerol.
[434] In order to produce epothilone D in medium containing methyl oleate, 1 mL of frozen oil-applied cells was inoculated into 3 mL of CYE-MOM medium in glass culture test tubes. Cells were incubated at 30 ° C., 175 rpm for 2 days and transferred to a 250 mL Erlenmeyer flask containing 50 mL of CYE-MOM medium. The resulting seed cultures were further incubated for 2 days under the same conditions and inoculated into 50 mL production flasks at 5% (v / v) inoculation scale.
[435] To prepare the inoculation for 5 L-fermentation, 25 mL of oil-adapted species were transferred to a 2.8 L Fernbach flask containing 475 mL of CYE-MOM medium. Cells were incubated for 2 days at 30 ° C. and 175 rpm. 250 mL of this secondary seed culture was then inoculated into a 5-L fermenter containing 4.75 L of production medium to obtain a final inoculation concentration of 5% (v / v).
[436] Shake flask production
[437] In the absence of methyl oleate, a batch culture of Myxococcus xanthus K111-40-1 was prepared as follows. One gram of XAD-16 resin (Rohn and Haas) was autoclaved at 121 ° C. for 30 minutes in a 250 mL Erlenmeyer flask containing 5 mL deionized water. Excess water was removed from the flask and 50 mL of CTS medium, pH 7.6, consisting of 5 g / L Cagiton, 2 g / L MgSO ₄ .7H ₂ O and 50 mM HEPES was added. When the autoclave treatment of the adsorption resin in the presence of the production medium binds the essential nutrients required by the cells, the resin and the media components were sterilized separately. 2.5 mL of the seed culture was inoculated into the production flask and incubated at 30 ° C. and 175 rpm for 6 days.
[438] Batch cultures were prepared as described above in the presence of methyl oleate. In addition, 10 mL / L concentrated H ₂ SO ₄ , 14.6 g / L FeCl ₃ · 6H ₂ O, 2.0 g / L ZnCl ₃ , 1.0 g / L MnCl ₂ · 4H ₂ O, 0.43 g / L CuCl _{2 4} mL / L of filter-sterilized microelement solution consisting of 2H ₂ O, 0.31 g / LH ₃ BO ₃ , 0.24 g / L CaCl ₂ 6H ₂ O, and 0.24 g / L Na ₂ MO ₄ 2H ₂ O 7 ml / L of methyloleate was reinforced. The production flask was inoculated with 2.5 mL of oil-adapted species culture and incubated at 30 ° C., 175 rpm for 5 days.
[439] A fed-batch culture intermittently supplied with Kaziton and methyl oleate was prepared as follows. One gram of XAD-16 resin was autoclaved at 121 ° C. for 30 minutes in a 250 mL Erlenmeyer flask with 5 mL deionized water. After sterilization, excess water was removed from the flask and 2 mL / L methyl oleate, 4 mL / L trace element solution, and 50 mL of CTS medium at pH 7.6 supplemented with 50 mM HEPES were added. The production flask was inoculated with 2.5 mL of oil-adapted species culture and incubated at 30 ° C., 175 rpm. Two days after the inoculation, the culture medium was added with 2 g / L Cajiton and 3 mL / L methyl oleate at 24 hour intervals. The Kaziton feed was prepared as a concentrated 100 g / L solution. Cultures were incubated for 10-12 days until substantial cell lysis was observed.
[440] All production cultures can be grown to 500 mL in 2.8-L Fernbach flasks under the same culture conditions.
[441] Fermenter production
[442] [283] A 5-L scale fed-batch fermentation with an intermittent or continuous supply of kaziton and methyl oleate was prepared as follows. 20 g / L XAD-15 and 2 g / LMgSo ₄ .7H ₂ O were autoclaved for 30 minutes at 121 ° C. in a 5-L fermenter (B.Braun) containing 4.75 L of deionized water. After sterilization, concentrated kaziton solution (150 g / L), methyl oleate and trace elements were aseptically added to the bioreactor so that the final concentrations of kaziton, methyl oleate and trace elements were 5 g / L, 2 mL / L and It was set to 4 mL / L. The medium was then inoculated with 250 mL of oil-adapted species. Fermentation was carried out at 30 ° C. with a ventilation rate of 0.4-0.5 v / v / m and an initial stirring speed of 400 rpm. Dissolved oxygen was adjusted to 50% saturation by stirring the cascade at 400-700 rpm. Culture pH was maintained at 7.4 by automated addition of 2.5N KOH and 2.5MH ₂ SO ₄ . Twenty four hours after inoculation, kaziton (150 g / L) and methyl oleate were added to the production medium at a rate of infusion of 2 g / L / 1 day of cartonage and of 3 mL / L / 1 day methyl oleate. The injection was delivered as a single bolus every 24 hours or continuously using a peristaltic pump (W. Marlow). Cells were incubated for 10-12 days until cell lysis was recognized at significant levels.
[443] Epothilone Quantitation
[444] Prior to using XAD-16 resin for fermentation, 1 mL of culture broth was taken from the production flask or bioreactor and centrifuged at 13,000 g for 10 minutes. Quantification of the epothilone product in the supernatant using Hewlett Packard 1090 HPLC by UV detection at 250 nm. 500 microliters of supernatant were injected through a 4.6 × 10 mm guard column (Inertsil, ODS-3, 5 μm). Online extraction was performed with 100% water at a flow rate of 1 mL / 1 minute for 0.5 minutes, followed by 1.5 minutes using a 50% acetonitrile gradient. The first 2 minutes were bypassed the eluate to be sent as it was and then passed through a longer separation column (4.6 × 150 mm, Inertsil, ODS-3, 5 μm). Separation of epothilones C and D was carried out for 8 minutes using a gradient from 50% to 100% acetonitrile followed by washing with 100% acetonitrile for 3 minutes. Under these conditions, epothilone C eluted at 9.4 minutes and epothilone D at 9.8 minutes.
[445] Using XAD-16 resin, 5-50 mL of well mixed culture broth and resin were collected from the production flask or bioreactor. After the resin was fixed by gravity, the culture broth was decanted. The resin was washed with 5-50 mL of water and again fixed by gravity. The aqueous mixture was completely removed and the epothilone product was extracted from the resin with 100% methanol. The amount of solvent used was 50% of the sample volume. Epothilones C and D were quantified by HPLC analysis with UV detection at 250 nm. 50 microliters of methanol extract were injected through two 4.6 × 10 mm guard columns (Inertsil, ODS-3,5 μm) and a longer 4.6 × 150 mm guard column (Inertsil, ODS-3,5 μm). The assay was isocratic and eluted for 18 minutes using 60% acetonitrile and 40% water at a flow rate of 1 mL / 1 min. Under these conditions, epothilone C was detected at 10.3 minutes and epothilone D at 13.0 minutes. Standards were prepared using purified Epothilone D.
[446] Cell growth measurement
[447] [286] Cell growth in the absence of methyl oleate was monitored by measuring optical density (OD) at 600 nm. Samples were diluted with water until the final OD value was less than 0.4. Since the addition of methyl oleate to the medium formed an emulsion exhibiting strong absorption at 600 nm, cell growth in the presence of methyl oleate was measured by dry cell weight (DCW). 40 millimeters of culture broth was centrifuged at 4200 g for 20 minutes in pre-weighed test tubes. The pellet was then washed with 40 mL of water and dried at 80 ° C. for 2 days prior to weighing.
[448] Ammonia measurement
[449] [287] One milliliter of fermentation broth was clarified by centrifugation at 13,000 g for 5 minutes. This supernatant was used for ammonia analysis using the ammonia assay kit (Sigma). Samples were diluted with 20-100 times water until the final concentration was below 880 μM.
[450] Methyl oleate measurement
[451] The remaining methyl oleate in the 1-mL fermentation broth was extracted with 5 mL of acetonitrile. The mixture was stirred with vortex and clarified by centrifugation at 4200 g for 20 minutes. Quantification of methyl oleate was performed by HPLC analysis with UV detection at 210 nm. 50 microliters of supernatant were injected through two 4.6 × 10 mm guard columns (Inertsil, ODS-3,5 μm) and a longer 4.6 × 150 mm guard column (Inertsil, ODS-3,5 μm). The column was washed with acetonitrile-water (1: 1) at a flow rate of 1 mL / 1 min for 2 minutes. It was then eluted with a gradient of 50% to 100% acetonitrile for 24 minutes and then washed with 100% acetonitrile for 5 minutes. Because of the different carbon chain lengths of commercially available methyl oleate, this compound eluted as two main peaks detected at 25.3 minutes and 27.1 minutes. Methyl oleate bound to the XAD-16 resin was quantified from the methanol extract using the same HPLC method. A standard of methyl oleate was prepared in 83.3% acetonitrile. The consumption of methyl oleate by the cells was calculated as follows: (total methyl oleate added)-(residue methyl oleate in medium)-(methyl oleate bound to resin).
[452] result
[453] [289] 5g / L Kaji tone (pancreatic casein hydride) and by using a simple production medium consisting only of magnesium sulfate in 2g / L and cultured by batch fermentation of Rhodococcus mikso glass tooth K111-40-1 strain for the first time. The basic performance of these cells is shown in FIG.
[454] [290] Maximum cell density and epothilone D production were obtained at OD ₆₀₀ 1.6 and the corresponding titer 0.16 mg / L after 3 days inoculation. Since then, both cell density and production yield have actually decreased. As the cells consumed kaziton, a gradual accumulation of ammonia in the production medium was also detected. At the end of the five day fermentation, the final concentration of ammonia reached 20 mM.
[455] Effect of XAD-16 on Production Stability
[456] In order to prevent rapid decomposition of epothilone D, hydrophobic adsorption resin XAD-16 was added to the production medium to bind and stabilize the discharged product. XAD-16 is a polyaromatic resin that has already been used by Gerth et al. (1996) to separate epothilones A and B from fermentation of microbial producer Soranium cellulose So ce90. As shown in FIG. 7, the presence of the adsorptive resin had no effect on cell growth. However, this effectively reduced the loss of epothilone D in the fermentation broth, which increased the recovery of this product by three times.
[457] Badge development
[458] [292] In an effort to develop media that can support higher cell densities and epothilone productivity, the effect of the kaziton on growth and production yields was from 1 g / L to 40 g / L in the production medium. Was evaluated by changing. Although cell growth was also promoted by increasing the carton concentration, the specific productivity of the cells decreased significantly as shown in FIG. 8. The optimum Cagtone concentration for epothilone D production was 5 g / L, with higher concentrations reducing titers.
[459] Because the enhancement of media was limited by the use of casinoton, another substrate was evaluated as a reinforcement for basal production batching. Screening various oils in detail, it was identified that methyl oleate is the carbon source that promoted the greatest increase in epothilone D production, summarized in Table 3.
[460] Oil (7mL / L)Relative productivity of epothilones for non-oil supplemented controls (%) Methyl oleate780 Ethyl oleate740 Coconut oil610 Laad470 Profile oleite420 Sesame oil380 Glycerol tri-oleate370 Salad Oil360 Sunflower oil330 Soybean oil290 Methyl heptadecanoate190 No oil (control)100 Methyl Nonadecanoate96 Methyl pelargonate40 Rapeseed oil40
[461] [294] However, direct cell lysis occurs when methyl oleate is added directly to the production medium. Thus, the cultivation was incubated in the presence of methyl oleate prior to production fermentation. Interestingly, this adaptation process made the cells less sensitive to lysis. As shown in Table 4 (growth and productivity improvement compared to baseline performance in CTS medium in batch fermentation), 3.3 mg of epothilone D titer with 7 mL / L of methyl oleate concentration and 4 mL / L of trace element concentration. / L and a peak biomass concentration of 2.1 g / L were obtained.
[462] Fermentation conditionMax Cell Density (g DCW / L)Maximum Epothilone D Production (mg / L) XAD-16 Free CTS Badge, Batch Process0.44 ± 0.040.16 ± 0.03 XAD-16 Added CTS Medium, Batch Process0.44 ± 0.040.45 ± 0.09 CTS medium with 7 mL / L methyl oleate, batch process1.2 ± 0.10.12 ± 0.02 CTS medium with 7 mL / L methyl oleate and 4 mL / L trace elements, batch process2.1 ± 0.23.3 ± 0.7 Intermittent Fed-Batch Process0.6 ± 0.69.8 ± 2.0 Continuous Fed-Batch Process7.3 ± 0.723 ± 4.6
[463] As shown in FIG. 9, no further titer enhancement was observed at higher methyl oleate concentrations.
[464] [296] In addition to demonstrating the significance of methyl oleate on cell growth and production, the graphs above also emphasize the importance of trace elements in the production medium. It was anticipated that the nutrients supplied by Cazyton would not be sufficient for vigorous cell growth, adding trace metals from outside with methyl oleate. Surprisingly, this supply has been found to be essential for both growth and production growth. That is, in the absence, the maximum biomass concentration and epothilone D titer were only 1.2 g / L and 0.12 mg / L, respectively. This low titer was comparable to that obtained in basal medium.
[465] Fed-Batch Development
[466] In the presence of optimal concentrations of methyl oleate and trace elements in the batch fermentation process, logarithmic growth of myxococcus xanthus strains occurred during the first two days of inoculation. Production of epothilone D started with normal phase onset as shown in FIGS. 10A and 10B and cell lysis occurred with depletion of methyl oleate on day 5. The time course of methyl oleate consumption and ammonia generation is also shown. The concentration of ammonia in the production medium was <4 mM through the fermentation process.
[467] In order to extend the production period of the cells in the flask medium, kaziton and methyl oleate were injected into the medium once a day at 2 g / L 1 day and 3 mL / L / 1 day, respectively. Substrate infusion was initiated 48 hours after inoculation and the cells grew logarithmically for 3 days thereafter. As shown in FIG. 11A, the biomass concentration remained constant on day 5 and reached a maximum of 6.3 g / L on day 10. Again, the production of epothilone D was consistent with the normal phase, reaching a final yield of 9.8 mg / L at the end of day 12. As shown in FIG. 11B, both consumption of methyl oleate and generation of ammonia increased at a constant rate throughout the fermentation process.
[468] Methyl oleate was depleted at the same rate as injected into the bioreactor, and ammonia accumulated at a rate of 3.2 mM / 1 day. Lower infusion rates of cajiton or methyl oleate reduced titer of epothilone D, while higher infusion rates resulted in early lysis of cells before significant production was achieved.
[469] [300] To test the efficiency of the fed-batch process at larger scales, kaziton and methyl oleate were added intermittently at 24-hour intervals on a 5 L-fermentation scale in a bioreactor. As shown in FIG. 12, the production curve obtained was very similar to that of the flask culture. Substrate infusion was started 24 hours after inoculation and production of epothilone was started on day 4. 9.2 mg / L of peak epothilone D titer was obtained 10 days after inoculation.
[470] [301] To assess the effect of more sophisticated injection techniques on growth and productivity, dual feeds were delivered continuously to the bioreactor. As shown in FIG. 13A, the performance of the continuous infusion had no effect on cell growth, but increased its productivity almost threefold. Final epothilone D titer 27 mg / L was obtained 10 days after inoculation. As shown in FIG. 13B, methyl oleate was consumed at the same rate as it was added to the production medium and ammonia was released at a constant rate of 6.4 mM / day over the fermentation period.
[471] discussion
[472] [302] Although epothilone D has recently been chemically synthesized (Harris et al., 1999; Meng et al., 1998; Sinha et al., 1998), the complex 20-step process is not economically suitable for large-scale production of this compound. none. While the initial yield of epothilone D in the Myxococcus xanthus strain was 0.16 mg / L, the improved fermentation process of the present invention substantially increased the production level to 23 mg / L.
[473] [303] One of the important hurdles in obtaining higher epothilone D titers was that the product degraded too quickly in the fermentation broth. This problem was alleviated by incorporating the adsorptive resin into the culture medium. The addition of XAD-16 had no effect on cell growth, but rather the loss of secreted product was minimized and its recovery increased by three times.
[474] [304] Another obstacle to increasing production yields is the limited titer improvement when using kaziton as the main carbon and nitrogen source. Although the growth of myxococcus xanthus strains is promoted by increasing the concentration of kaziton, the titer decreases dramatically when the concentration exceeds 5 g / L. The inhibitory effect of high levels of peptone or cajiton on this secondary metabolite productivity has already been demonstrated in several other myxobacteria fermentations, which is believed to be due to the accumulation of ammonia in the culture medium.
[475] [305] Myxococcus xanthus strains do not metabolize polysaccharides and sugars (Reichenbach and Dworkin, 1991), so their ability to use oil as a carbon source has been tested. The oxidation of fatty acids not only acts as an energy source in the cell, but the formation of acetyl-CoA as a degradation product also provides a precursor of epothilone biosynthesis, making oil an attractive carbon source. When fermentation of Saccharopolyspora erythraea, Streptomyces fradiae and Streptomyces hygroscopicus were added, the oils were added to fermentation of erythromycin, tylo , respectively. Production of polyketide molecules such as L-683590, a renal and immunomodulatory agent, has been shown to be enhanced (Mirjalili et al., 1999; Choi et al., 1998; Junker et al., 1998).
[476] [306] Screening of various oils identified methyl oleate as a leading candidate for promoting cell growth and epothilone D productivity. This improvement, however, was only observed when co-adding trace metals to the production medium. When only methyl oleate alone was added in an amount of 7 mL / L, the maximum cell density increased from 0.4 g DCW / L to 1.2 g DCW / L, while production remained at baseline levels. When trace elements were fed from the outside in an amount of 4 mL / L, the peak biomass concentration increased to 2.1 g DCW / L, and the epothilone D titer jumped from 0.45 mg / L to 3.3 mg / L. These findings indicate that nutritional deficiencies in kaziton may be important for growth and epothilone formation of Myxococcus xanthus strains.
[477] [307] In order to establish the optimal concentration of methyl oleate and trace elements in the batch process, efforts were made to develop injection operations to extend production periods while maintaining robust cell growth. Evaluating the results of intermittent and continuous infusion of kaziton and methyl oleate at a constant rate, both methods showed a similar improvement in growth profile. At optimal infusion of both substrates, a maximum cell density of about 6.8 g DCW / L was obtained. In both cases, methyl oleate was depleted as it was added to the fermentation medium.
[478] [308] In contrast to cell growth and methyl oleate consumption, epothilone growth and ammonia generation were strongly influenced by the choice of infusion strategy. In continuous fed-batch cultures, the peak epothilone D titers were 23 mg / L. This is almost 2.5 times the titer obtained for intermittent fed-batch cultures. Using continuous infusion, the rate at which ammonia was released by the cells was also twice as high, indicating that the consumption of cartons was that fast. With this. These results indicate that low titers in intermittent ped-batch processes may be due to catabolite repression or not due to ammonia accumulation. In addition, these results suggest that the productivity of myxococcus xanthus strains is sensitive to the amount of substrate present in the culture medium and can be maximized in substrate-limiting conditions. This is also in agreement with the observation that increasing the concentration of cathetone in the production medium results in higher cell density but lower titer.
[479] [309] Compared to batch culture using basal medium, in continuous fed-batch culture, cell density was increased 17 times and titer was increased 140 times. This process was performed on a 5-L scale on a 1000-L scale and in the same way. The results shown in connection with the production of epothilone D in accordance with the production scale gives jeuae type the possible use of the strain as a host mikso Lactococcus glass tooth in the production of other biologically active molecules of mikso bacteria.
[480] Reference
[481] [310] Bollag et al. 1995. Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res 55: 2325-2333.
[482] [311] Choi et al. 1998. Effects of rapeseed oil on activity of methylmalonyl-CoA carboxyltransferase in culture of Streptomyces fradiae. Biosci Biotechnol Biochem 62: 902-906.
[483] [312] Chou et al. 1998. Desoxyepothilone B: An efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B. Proc Natl. Acad Sci USA 95: 9642-9647.
[484] [313] Gerth et al. 1996. Epothilons A and B: Antifungal and cytotoxic compounds from Sorangium cellulosum (myxobacteria) -production, physico-chemical, and biological properties. J Antibiot (Tokyo) 49: 560-563.
[485] [314] Gerth et al. 2000. Studies on the biosynthesis of epothilones: the biosynthetic origin of the carbon skeleton. J Antibiot (Tokyo) 53: 1373-1377.
[486] [315] Gerth et al. 2001. Studies on the biosynthesis of epothilones: The PKS and epothilone C / D monooxygenase. J Antibiot (Tokyo) 54: 144-148.
[487] [316] Harris et al. 1999. New chemical synthesis of the promising cancer chemotherapeutic agent 12, 13-desoxyepothilone B: Discovery of a surprising longrange effect on the diastereoselectivity of an aldol condensation. J Am Chem Soc 12: 7050-7062.
[488] [317] Hecht et al. 1990. Hollow fiber supported gas membrane for in situ removal of ammonium during an antibiotic fermentation. Biotechnol Bioeng 35: 1042-1050.
[489] [318] Junker et al. 1998. Use of soybean oil and ammonium sulfate additions to optimize secondary metabolite production. Biotechnol Bioeng 60: 580-588.
[490] [319] Kowalski et al. 1997. Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel. J Biol Chem 272: 2534-41.
[491] [320] Meng et al. 1997. Remote effects in macrolide formation through ringforming olefin metathesis: An application to the synthesis of fully active epothilone congeners. J Am Chem Soc 119: 2733-2734.
[492] [321] Mirjalili et al. 1999. The effect of rapeseed oil uptake on the production of erythromycin and triketide lactone by Saccharopolyspora enythraea . Biotechnol Prog 15: 911-918.
[493] [322] Molnar et al. 2000. The biosynthetic gene cluster for the microtubulestabilizing agents epothilones A and B from Sorangium cellulosum So ce 90. Chem Biol 7: 97-109.
[494] [323] Reichenbach et al. The Prokaryotes II Eds. New York: Springer-Verlag. p 3417-3487.
[495] [324] Reichenbach et al. 1993. Production of Bioactive Secondary Metabolites. In Dworkin M, Kaiser D, editor. Myxobacteria II. Washington, DC: American Society for Microbiology. p 347-397.
[496] [325] Sinha et al. 1998. The antibody catalysis route to the total synthesis of epothilones. Proc Natl Acad Sci USA 95: 14603-14608.
[497] [326] Su et al. 1997. Structure-activity relationships of the epothilones and the first in vivo comparison with paclitaxel. Angew Chem Int Ed Engl 36: 2093-2096.
[498] [327] Tang et al. 2000. Cloning and heterologous expression of the epothilone gene cluster. Science 287: 640-642.
[499] B. Production of Epothilone B
[500] flask
[501] [328] A 1 mL vial of K111-32-25 strain was thawed and the contents transferred to 3 mL CYE seed media in glass test tubes. The cultures were incubated at 30 ° C. for 72 ± 12 hours and then 3 mL of this test tube culture was subcultured in 50 mL of CYE medium in a 250 mL Erlenmeyer flask with baffle. This CYE flask was incubated at 30 ° C. for 24 ± 8 hours, using 2.5 mL of this seed (5% v / v) epothilone producing flask (250 mL of CTS-TA medium in a 250 mL baffle flask with baffle). ) Was inoculated. The flasks were incubated at 30 ° C. for 48 ± 12 hours with medium starting from pH 7.4.
[502] Fermenter
[503] [329] A similar inoculation expansion of K111-32-25 as described above was used, except that 25 mL of 50 mL CYE seeds were subcultured to 500 mL CYE. This secondary seed was inoculated into a 10 L fermenter containing 9.5 L CTS-TA and 1 g / L sodium pyruvate. The process variable step points for this fermentation are: pH-7.4; Stirring speed-400 rpm; Sparge speed-0.15 vvm. These variables were sufficient to keep the DO above 80% saturation. pH adjustment was performed by adding 2.5N sulfuric acid and sodium hydroxide to the culture. Peak epothilone titers were obtained at 48 ± 8 hours.
[504] C. Production of Epothilone D
[505] flask
[506] [330] K111-32-25 strain (described in Example 4) 1 mL vials were thawed and the contents transferred to 3 mL CYE seed media in glass test tubes. The cultures were incubated at 30 ° C. for 72 ± 12 hours and then 3 mL of this test tube culture was subcultured in 50 mL of CYE medium in a 250 mL Erlenmeyer flask with baffle. The CYE flasks were incubated at 30 ° C. for 24 ± 8 hours, using 2.5 mL of this seed (5% v / v) epothilone producing flask (50 mL of 1 × flour gluten medium in 250 mL baffle flasks with baffles). ) Was inoculated. The flasks were incubated at 30 ° C. for 48 ± 12 hours with medium starting from pH 7.4.
[507] Fermenter
[508] A similar inoculation expansion of K111-40-1 as described above was used, except that 25 mL of 50 mL CYE seeds were subcultured to 500 mL CYE. 250 mL of this secondary seed was used to inoculate a 5 L fermenter containing 4.5 L CTS-TA and 1 g / L sodium pyruvate. The process variable step points for this fermentation are: pH-7.4; Stirring speed-400 rpm; Sparge speed-0.15 vvm. These variables were sufficient to keep the DO above 80% saturation. pH adjustment was performed by adding 2.5N sulfuric acid and sodium hydroxide to the culture. Peak epothilone titers were obtained at 36 ± 8 hours. The peak epothilone C titer was 0.5 mg / L and the peak epothilone D titer was 1.6 mg / L.
[509] [332] The medium used in Table 5 and the components thereof are summarized.
[510] CYE species ingredient densityCasino10 g / L Yeast Extract (Difco)5 g / L MgSO ₄ 7H ₂ O (EM Science)1 g / L HEPES buffer50 mM CTS-TA Production Medium ingredient densityCasino (Difco)5g / L MgSO ₄ 7H ₂ O (EM Science)2 g / L L-alanine, L-serine, glycine1 mg / L HEPES buffer50 mM 1x Wheat Flour Gluten Production Medium ingredient densityWheat Flour Gluten (Sigma)5g / L MgSO ₄ 7H ₂ O (EM Science)2 g / L HEPES buffer50 mM
[511] CYE seed medium and CTS0TA production medium were sterilized with autoclave at 121 ° C. for 30 minutes. Flour gluten production medium is sterilized with autoclave at 121 ° C. for 45 minutes.
[512] D. Myxocaucus XanthusEpothilone C and D Production from
[513] [333] In one aspect, the invention provides improved fermentation methods in mikso Rhodococcus strains, including Lactococcus glass K111-40-1 tooth (but not limited to) mikso, to the fermentation medium is raised to ammonia in the method Provides a carbon source that can be used without work. In one preferred embodiment, the carbon source is an oil such as methyl oleate or similar oils. In shake flask tests with different injection ratios, this method produced epothilones C and D, mainly producing epothilones at a concentration of 15-25 mg / L as described below.
[514] Cultivation
[515] [334] 2mL / L methyl oleate to lifted mikso Lactococcus glass tooth K111-40-1 cells frozen 1 mL vial was grown in 50mL reinforced CYE medium in sterile glass test tube, 1mL / L methyl oleate in a fresh CYE reinforced 3mL The medium was inoculated. This test tube was incubated for 24 hours on a shaker at 30 ° C., 250 RPM. The inoculum in the glass test tube was then transferred to a 250 mL baffle-free flask containing 50 mL fresh CYE medium supplemented with 2 mL / L methyl oleate. This flask was incubated for 48 hours at 30 ° C., 250 RPM shaker.
[516] Production flask
[517] [335] 1 g of Amberlite XAD-16 was sterilized by autoclaving for 30 minutes at 121 ° C. in a 250 mL-baffle-free flask. The flasks were inoculated with 5% (v / v) species and placed in an incubator shaker operating at 250 RPM, 30 ° C. Sterile methyl oleate 3 mL / L / 1 day infusion was initiated 2 days after inoculation, and the carton 2 g / L / 1 day infusion was initiated 1 day after inoculation.
[518] Product extraction
[519] After 14 days, the XAD resin in the production flask was transferred to a 5 mL centrifuge test tube. Excess media in vitro was drained and care was taken not to remove the resin at this time. The XAD resin was then placed in 25 mL of water and fixed. The water in the test tube was decanted, taking care not to remove the resin and 20 mL of methanol was added to the test tube. Centrifuge tubes were placed in a shaker at 175 RPM for 20-30 minutes to extract the epothilone product from the resin. The methanol extract was transferred to fresh centrifuge test tubes for storage and subjected to LC / MS analysis.
[520] [337] The medium used in Table 6 and its components are summarized.
[521] CYE species ingredient densityCasino10 g / L Yeast Extract (Difco)5 g / L MgSO ₄ 7H ₂ O (EM Science)1 g / L Production badge ingredient densityCasino (Difco)5g / L MgSO ₄ 7H ₂ O (EM Science)2 g / L After autoclave, added to production medium 1000x Trace Element Solution4 mL / L Methyl oleate2 g / L
[522] CYE medium and production medium were sterilized by autoclaving at 121 ° C. for 30 minutes. The trace element solution was filter-filtered and methyl oleate was autoclaved separately.
[523] All of the components in the following Table 7 were added to a 10 mL / L concentrated sulfuric acid solution to combine to a final volume of 1 L to form a trace element solution.
[524] 1000x Trace Element SolutioningredientdensityFeCl ₃ 8.6 g / L ZnCl ₂ 2.0 g / L MnCl ₂ 4H ₂ O1.0 g / L CuCl ₂ · 2H ₂ O0.43 g / L H ₃ BO ₃ 0.31 g / L CaCl ₂ · 6H ₂ O0.24 g / L Na ₂ MoO ₄ 2H ₂ O0.24 g / L
[525] The obtained solution was filter filtered.
[526] E. Myxocaucus Xanthus, Production and purification of epothilones from
[527] Myxocaucus XanthusStrain description
[528] Strain K111-25-1 is an epothilone B producing strain, which also produces epothilone A. Strain K111-40-1 also produces epothilone C as an epothilone D producing strain.
[529] On the plate Myxocaucus XanthusMaintenance of
[530] [340] Myxococcus xanthus strains were maintained on CYE agar plates (see Table 8 for plate composition). Colonies appeared about 3 days after streaking on the plate. Plates were incubated at 32 ° C. until desired growth levels were stored and then stored for 3 weeks at room temperature (cells could die if stored at 4 ° C. on plates).
[531] CYE Agar Plate *ingredientdensityHydrolyzed casein (pancreatic digest)10 g / L Yeast extract5 g / L Agar15 g / L MgSO ₄ 1 g / L 1 M MOPS buffer solution (pH 7.6)10 mL / L
[532] * 1 L agar medium batch was autoclaved for 45 minutes and then poured into Petri dishes.
[533] Oil adaptation of myxococcus xanthus for cell bank
[534] [341] Non-oil adapted colonies from frozen cell vials or CYE plates were transferred to a 50 mL glass culture tube containing 3 mL of CYE species medium and one drop of methyl oleate from a 100 μL pipette. Cells were incubated for 2-6 days (30 ° C., 175 rpm) until the culture was observed to be compact under the microscope. Since these cells do not always adapt well to oil, they started with a few (5-7) test tubes.
[535] Cell Bank Manufacturing Method (Master Cell Bank)
[536] [342] An oil-adapted in vitro culture was initiated as described above. Once the in vitro culture was sufficiently dense (OD = 5 +/- 1), 50 mL of CYE-MOM seed media (see table below for medium composition). The entire tube contents were transferred to a sterile 250 mL shake flask containing. After incubation for 48 ± 12 hours in a shake incubator (30 ° C., 175 rpm), 5 mL of this species culture was transferred to 100 mL of CYE-MOM in a 500 mL shake flask. This culture was continued for 1 day in a shake incubator (30 ° C., 175 rpm). It was observed under a microscope whether the culture was properly incubated or not contaminated.
[537] 80 mL of this species and 24 mL of sterilized 90% glycerol in a sterile 250 mL shake flask were mixed. Mix thoroughly, mix well, and dispense aliquots of 1 mL of this mixture into 100 sterile, pre-labeled frozen vials. The vials were slowly frozen in a -80 ° C freezer.
[538] How to Bond Cell Banks (Work Cell Banks)
[539] In vitro culture was initiated by thawing one of the prepared master cell bank vials to room temperature, and then the entire contents were transferred to a glass test tube containing 3 mL of CYE-MOM species medium. Once this test tube culture was sufficiently dense (OD = 5 +/- 1), the entire tube contents were transferred to a sterile 250 mL shake flask containing 50 mL of CYE-MOM seed medium. After incubation (30 ° C., 175 rpm) for 48 ± 12 hours, 5 mL of this species culture was transferred to 100 mL of CYE-MOM in a 500 mL shake flask. This culture was continued for 1 day in a shake incubator (30 ° C., 175 rpm). It was observed under a microscope whether the culture was properly incubated or not contaminated.
[540] 80 mL of this species and 24 mL of sterilized 90% glycerol in a sterile 250 mL shake flask were mixed. Mix thoroughly, mix well, and dispense aliquots of 1 mL of this mixture into 100 sterile, pre-labeled frozen vials. The vials were slowly frozen in a -80 ° C freezer.
[541] Composition of species
[542] [346] The same seed medium as described in Table 9 was used for cell bank preparation and amplification of cell bank vials to a given volume.
[543] CYE-MOM Species *ingredientdensityHydrolyzed Casein (pancreatic digest)-Difco10 g / LYeast Extract-Difco5 g / L _{MgSO 4 · 7H 2 O - EM} Science1 g / LMethyl Oleate-Cognis2 mL / L
[544] * Footnote: Methyl oleate does not mix completely with other ingredients because it forms an emulsion in kaziton and is added after all other ingredients have been added.
[545] Large inoculation for shake flasks, 5L, and 1000L fermentation
[546] [347] Frozen working cell bank vials of methyloleate adapted cells were thawed. The entire vial contents were transferred to a 50 mL glass culture tube containing 3 mL of CYE-MOM seed medium. This test tube was placed in a shaker (30 ° C., 175 rpm) and incubated for 48 ± 12 hours. The entire culture tube contents were transferred to a 250 mL shake flask containing 50 mL of CYE-MOM seed medium. The flask was placed in a shaker (30 ° C., 175 rpm) and incubated for 48 ± 12 hours. For use in shake flask experiments, this culture was amplified by subculture of 10 mL of this culture into 40 mL of fresh CYE-MOM in five new seed flasks. Flask Volume (30-100 mL) Seed flasks were incubated for 24 ± 12 hours on shaker (30 ° C., 175 rpm) to use the inoculum for production culture. Production flasks were inoculated with a combined initial volume of 4.5% (media and production medium).
[547] In order to prepare small-scale (5-10 L) fermented seeds, the entire contents of one of these 50 mL seed flasks were subcultured into a sterile 2.8 L Fernbach flask containing 500 mL of CYE-MOM. This Pervas flask was incubated for 48 ± 12 hours in shaker (30 ° C., 175 rpm) and used as fermentor inoculum. Production fermentation was inoculated at a scale of about 5% of the combined initial volume.
[548] [349] Large seed (1000L) fermentations require additional seed amplification. Here, 1 L of Fernbach flask seeds were used to inoculate a 10 L seed fermenter containing 9 L of CYE-MOM (5% by volume). The pH of the fermenter was adjusted to 7.4 by adding 2.5N potassium hydroxide and 2.5N sulfuric acid. The temperature was fixed at 30 ° C. Dissolved oxygen was maintained at a saturation of at least 50% by cascading the stirring rate between 400-700 rpm. Initial stirring speed was fixed at 400 rpm and sparging speed was maintained at 0.1 v / v / m. After incubation for 24 ± 12 hours in a 10 L fermenter, the entire culture was transferred to a 150 L fermenter containing 90 L CYE-MOM. The pH was again adjusted to 7.4 with 2.5N potassium hydroxide and 2.5N sulfuric acid. The temperature was fixed at 30 ° C. Dissolved oxygen was maintained at a saturation of at least 50% by cascading the stirring rate between 400-700 rpm. Initial stirring speed was fixed at 400 rpm and sparging speed was maintained at 0.1 v / v / m.
[549] XAD-16 Resin for Fermentation
[550] [350] The required amount of XAD-16 resin (Rohn & Haas) was placed in a methanol safety vessel having a volume of at least three times the weight of the XAD-16 resin (ie, 1.2 kg of resin required at least 3.6 L of vessel). The resin was thoroughly washed with 100% methanol to remove all monomers present in the new resin. Liter of methanol (ie 6 liters of methanol per 3 kilograms of XAD-16) corresponding to twice the kilogram weight of the resin was added. Methanol and XAD slurry were mixed for 5 minutes to remove any monomers present in XAD-16. The slurry was mixed with gentle stirring to minimize resin fractionation. Agitation was stopped and the resin was gravity fixed for at least 15 minutes. The methanol was drained from the vessel, leaving only a 0.5-1 inch layer of methanol on the XAD vegetation. XAD and methanol were transferred from the mixing vessel to an Amicon VA250 column. The upper bed support was attached to the column and the stopper was tightened clockwise to seal the bed support. The XAD in the column was washed with at least 5 column volumes of methanol at a rate of 300 ± 50 cm / hr. Methanol was collected in a waste solvent collection vessel. The XAD in the column was washed with at least 10 column volumes of deionized water at a rate of 300 ± 50 cm / hr.
[551] The composition of the epothilone production medium is shown in Table 10 below.
[552] CTS-MOm Production MediumingredientdensityCasino (Difco)5 g / LMgSO ₄ 7H ₂ O (EM Science)2 g / LXAD-1620 g / L Add after autoclave treatment 1000 x Trace Element Solution4 mL / LMethyl oleate2 mL / L
[553] * Footnote: Methyl oleate does not mix completely with other ingredients because it forms an emulsion in kaziton and is added after all other ingredients have been added. Trace element solutions are as described in Table 7.
[554] Preparation and Flask-Scale (50mL) Epothilone Production and Fermentation
[555] The resin was recovered by autoclaving 1 g of XAD-15 in a 250 mL shake flask with a sufficient amount of deionized water (˜3 mL). The flask was sterilized by autoclaving at 121 ° C. for 30 minutes. The following media components were aseptically added to the flasks: 50 mL of CTS-MOm production medium and 1 M HEPES buffer (titrated to pH 7.6 with potassium hydroxide) 2.5 mL of CYE seed flasks (4.5% volume / volume inoculation) Inoculation. Production flasks were incubated in shakers at 30 ° C. and 175 rpm.
[556] [353] The infusion of kaziton and methyl oleate was initiated 24 ± 6 hours after inoculation. At this point, and every 24 ± 6 hours thereafter, 1 mL of 100 g / L Cagiton solution and 150 μL of methyl oleate were injected. The infusion schedule was continued until 13 days after the initial infusion or until cells were observed to begin lysing (11-14 days). To determine the epothilone producing kinetics, a sample of mL of well mixed fermentation broth and XAD can be representatively sampled. In addition, a small (0.25-0.5 mL) sample of broth may be taken daily without XAD to visually examine the culture. If cytolysis is observed on a large scale, the rest of the culture volume should also be harvested.
[557] Preparation and Fermentation of 5-L Scale Epothilone Production
[558] [354] 100g XAD and 8g MgSO ₄ -7H ₂ O mix and with 3.9L of de-ionized water, was sterilized in a 5L B-Braun bioreactor (90 min, 121 ℃). Sufficient volume (133 mL) of Pasteurized Catonton / Deionized Water solution (150 g / L) was aseptically pumped to bring the final concentration of Cagiton in the fermentor to 5 g / L. An initial methyl oleate concentration of 2 ml / L is achieved by adding 10 mL of this oil. Finally, 16 mL of pre-sterilized microelement solution was aseptically added prior to inoculation. The fermenter was then inoculated with 200 mL of CYE species culture (4.8% volume / volume) and incubated for 24 ± 6 hours. At this point, infusions of cartonton (2 g / L / 1 day, continuous infusion) and methyl oleate (3 mL / L / 1 day total, semi-continuously every 90 minutes) were initiated. The airflow in the bioreactor was kept constant at 0.4-0.5 vvm (this change increases as the fermentation capacity increases with injection progress). Dissolved oxygen concentration was adjusted to 50% saturation by cascade stirring (400-700 rpm). The 100% saturated dissolved oxygen calibration point was established by fixing the initial stirring speed at 400 rpm and setting the initial airflow at 0.5 vvm. The pH was fixed at 7.4 by automatic addition of 2.5NH ₂ S ₄ and 2.5N KOH. Epothilone production continued for 11-14 days after inoculation, and bioreactors were harvested when cell lysis in broth samples became significant and the oxygen demand (indicated by the agitation rate) dropped sharply. The titer of epothilone D was generally 18-25 mg / L in this fermentation process.
[559] Fermentation and production of 1000L-scale epothilones
[560] [355] A 1000L fermenter for epothilone production was prepared as follows. 600 L of water and 18 L of XAD-16 (11.574 kg) were sterilized in a fermentor (45 min, 121 ° C.). Trace elements and MgSO ₄ were filtered directly (via pre-sterilized 0.2 micron polyethersulfone membrane capsule filters) into the fermentation vessel. Sufficient amount of concentrated MgSO ₄ solution (to a final concentration of 2 g / L in the fermentation bath) and 2.9 L of trace elements were added through the same capsule filter. Approximately 200 mL of a mixture of 117 g / L kaziton and 175 mL / L methyl oleate was sterilized in a 260 L injection tank. About 32 L of this sterile mixture was added to 1000 L fermenter. Water was filtered into a vessel (via the same capsule filter) to a final volume of 710 liters. Stir at 100 rpm. The back pressure was maintained at 100-300 mbar. When dissolved oxygen (DO) reached 50% after inoculation, the stirring speed was increased to 150 rpm. When the DO was 50% again, the stirring speed was increased to 200 rpm. DO was adjusted to 50% saturation by cascading airflow (0 Lpm-240 Lpm). The pH steppoint was maintained at 7.4 by auto addition of 2.5M KOH and 2.5NH ₂ SO ₄ . Fermenters were seeded with 38 L seeds from 150 L fermenters (5% volume / volume).
[561] [356] After the DO reaches 50% (second time, about 10 ± 5 hours after inoculation), the Kaziton-methyl oleate injection solution is added at a rate of 0.570 L / hour and the fermenter is harvested. The addition was continued. Bioreactor was harvested 10 days after inoculation. The final epothilone D titer was determined to be about 20 ± 5 mg / L.
[562] Fermentation Process
[563] For kinetic experiments in flasks, 5-50 mL of well mixed broth and XAD resin were collected with a 25 mL pipette and deposited in 10 mL or 50 mL conical test tubes. For bioreactor samples, samples of resin and mixed broth were deposited in 50 mL conical tubes. The conical tube was then allowed to stand for 10 minutes to keep the XAD from sinking to the bottom of the test tube. At this point the broth can be decanted from the XAD resin. If the XAD is not fixed, the broth can be removed using a 10-25 mL pipette.
[564] Methanol Extraction of XAD Resin for Epothilone Titer Proper
[565] After the XAD resin was gravity fixed at the bottom of the sample test tube (every sampling process), all of the supernatant was transferred to a new 50 mL conical test tube. The water was added to the 50 mL scale again to wash the XAD resin once, invert and mix thoroughly to allow the XAD resin to be gravity fixed. The aqueous mixture was decanted from the test tube while preventing XAD from pouring out. The last few mLs of water can be removed by inserting the tip of the 1 mL Pipetteman into the bottom of the test tube. Methanol was added to the test tube 25 mL scale and the test tube was blocked. Conical test tubes were placed horizontally on a shaker (20-30 ° C.) for 30 minutes to allow for complete extraction of all epothilones from the XAD resin.
[566] HPLC process for quantifying epothilones
[567] Epothilones C and D were analyzed using Hewlett Packard 1090 HPLC with UV detection at 250 nm. Methanol-extracted solution from XAD resin (50 μL) was added to a 4.6 × 10 mm guard column (Inertsil, C18 OD 53, 5 μm), and a longer column (4.6 × 150 mm) made of the same material for chromatographic separation. Injected through. This method, run for 18 minutes, isocratic in 60% acetonitrile and 40% water. By this method, epothilone D eluted at 13 minutes and epothilone C at 10.3 minutes. Standards were prepared using epothilone D purified from fermentation broth.
[568] Dry cell weight process for growth curve
[569] [34] The temperature of the Sorvall RC5B centrifuge (with SH-3000 bucket rotor) was fixed at 20 ° C. 50 mL conical test tubes weighed in an oven at 80 ° C. for at least one day were weighed. Tare weights were recorded and fermentation samples on the test tube side were identified. 40 mL broth (containing no XAD) was placed or pipetted into the tare test tube. The conical test tube was spun at 4700 RPM (4200 g) for 30 minutes. After sedimentation, the supernatant was decanted, the cell pellet was resuspended in 40 mL of deionized water, and the tube was placed in a drying oven at 80 ° C. for at least 2 days. The test tube was weighed and the final weight recorded in the test tube. The dry cell weight (DCW) can be calculated according to the following equation:
[570] DCW (g / L) = (final tube weight (g)-tare test tube weight (g)) / .04 L
[571] Measurement of Ammonium Ion Concentration
[572] In fermentation broth, ammonia concentrations in fermentation broth are usually measured. 1 mL of fermentation broth was clarified by centrifugation (5 min, 12000 rpm) using a microcentrifuge. Sigma's ammonia assay kit (Catalog # 171-UV) was used to quantify and was clarified by replacing fermentation broth instead of plasma as described in the kit protocol. Since the linear response range of this calorimetry was only 0.01176-0.882 mmoles / L, the purified fermentation samples were usually diluted 20-100 fold in deionized water to analyze the ammonium concentration within this range.
[573] Measurement of residual methyl oleate concentration
[574] The amount of residual methyl oleate present in the fermentation broth was evaluated by methanol extraction of the fermentation broth samples, followed by HPLC treatment of these extracted broth samples. Quantification of methyl oleate concentration was performed using hewlett Packard 1090 HPLC with UV detection at 210 nm. The whole broth sample (1-4 mL) was extracted with equal volume of methanol and centrifuged at 12,000 g to settle any insoluble components. The clarified supernatant (50 μL) was injected onto a 4.6 × 10 mm extraction column (Inertsil, C18 OD 53,5 μm), washed for 2 minutes with 50% acetonitrile and then the main column (4.6 × 150 mm, same normal phase and Flow rate), eluting with a gradient starting with 50% acetonitrile and finishing with 100% acetonitrile for 24 minutes. 100% acetonitrile column flow was maintained for 5 minutes. Due to its heterogeneous nature, methyl oleate is eluted as several discrete peaks, instead of a single pure compound. However, about 64-67% of the total methyl oleate extractable appeared as two major peaks eluting at 25.3 ± 0.2 minutes and 27.1 ± 0.2 minutes, respectively. Methyl oleate in a methanol extracted fermentation sample can be assessed by quantifying the total immunity of these two peaks and then extrapolating them to the sum of the area of these two peaks in the methyl oleate standard prepared in 50% water / methanol solution. .
[575] Purification and Crystallization of Epothilone D
[576] The present invention provides methods for the purification of epothilones and epothilones D and highly purified preparations of epothilones D, including crystalline form of epothilones D. The advantages of the process of the present invention include the need for only alcohol (such as methanol) and water, thereby providing an initial purification process that allows for the effective availability of the product pool while minimizing the need for time-consuming and labor-intensive evaporation steps. Can be mentioned. The process of the present invention requires only a single evaporation step, which requires evaporation of 1 L of ethanol every 10-15 g of epothilones. In this method, a column packed with a synthetic polystyrene-divinylbenzene resin such as HP20SS can be used to remove both polar and lipophilic impurities. This column generates intermediates containing 10% epothilone and also eliminates the need for liquid / liquid extracts that are very flammable or require the use of toxic solvents.
[577] [364] Another advantage relates to the use of 40-60 micron particle distributions and C18 resins, such as Bakerbond C18, which allows the use of low pressure columns and pumps (less than 50 psi), which greatly reduces the cost. The starting material for the C18 chromatography treatment is a solution loaded in dilute loading solvent. The solvent is sufficiently dilute so that the epothilone sticks in a tightly concentrated form, tight bands on top of the column, which allows the column to perform well under heavy loading (2-5 g epothilone / L resin). Chromatography is usually the most expensive step of the purification process, since typical column loadings are below 1 g / l, and these improvements provide significant cost savings. In addition, the process of the present invention allows the use of alcohols such as methanol in place of acetonitrile in the chromatography step. The pools containing epothilones were crystallized from a double solvent system (water is a forced solvent that provides the epothilone in crystalline form).
[578] In one embodiment, the purification method consists of the following steps and materials. The XAD resin in the fermentation broth is collected in (1) a filter basket, (2) eluted to give an XAD extract, (3) diluted with water and then (4) passed through an HP20SS column to obtain HP20SS pools. The HP20SS pool is diluted with (5) water and (6) treated with C18 chromatography to obtain an epothilone pool, which is diluted with (7) water and then (8) solvent exchanged to obtain a concentrated epothilone pool. This concentrated epothilone pool is (9) charcoal filtered, (10) evaporated and (11) crystallized to give a highly purified product.
[579] A total of 11 g of epothilone D was isolated and purified to obtain white crystalline powder from two 1000-L myxococcus xanthus fermentation processes (103100K and 1117001K). The purity of the final product was> 95% and the recovery of epothilone D was 71%.
[580] Table 11 below summarizes the HPLC methods used during the purification period.
[581] Epo1 method columnInertsil ODS3, 5 μm, 4.6 x 150 mmFlow rate1 ml / minColumn oven50 ℃Execution time15 minutesdetectionUV at 250 nmgradient0 minutes; 60:40 ACN / H ₂ O 12 min; 100: 0 ACN / H ₂ O 12.1 min; 60:40 ACN / H ₂ O Epo89 method columnInertsil ODS3, 5 μm, 4.6 x 150 mmFlow rate1 ml / minColumn oven50 ℃Execution time5 minutesdetectionUV at 250 nmgradient0 minutes; 78:22 ACN / H ₂ O
[582] [368] The materials used in this section are: HP20SS resin was purchased from Mitsubishi. The C18 resin was 40 g of C18 from Bakerbond and methanol was purchased from Fisher Bulk (55 gal). Deionized water was used.
[583] Fermentation carried out 1031001K
[584] First Step XAD Dissolution (K125-173)
[585] 17 liter (17 L) of XAD-16 resin was filtered from the fermentation broth using a Mainstream filtration unit with a 13-liter 150 μm capture basket. The captured XAD resin was packed into an Amicon VA250 column and washed with 1.0 L / min of 65 L (3.8 column volume) of water. The epothilone D product was eluted from the resin with 230 L of 80% methanol in water.
[586] Second Stage Solid Phase Extraction (K125-175)
[587] The loading solvent was diluted with 60 ^ methanol in water by adding 77 liters (77L) of water to the product pool (230L) of the first step. The resulting suspension (307 L) was mixed and loaded onto an Amicon VA180 column filled with 5 L of HP20SS resin previously equilibrated with 5 column volumes of 60% methanol. The loading flow rate was 1 L / min. After loading, the column was washed with 13 L of 60% methanol and eluted with 77 L of 75% methanol at a flow rate of 325 mL / min. 31 2.5 L-fractions were collected. Fractions 10-26 (42.5L) were found to contain epothilone D and these fractions were collected together.
[588] Third Step Chromatography (K125-179)
[589] The product pool of the second step was evaporated into oil using two 20-L rotovaps. During evaporation, ethanol had to be added to minimize foaming. The dry material was resuspended in 1.0 L of methanol and diluted with 0.67 L of water to make 1.67 L of 60% methanol solution. The resulting solution was loaded onto a 1-L C18 chromatography column (55 × 4.8 cm) previously equilibrated with 3 column volumes of 60% methanol. The loading flow rate averaged 64 mL / min. The loaded column was washed with 1 liter of 60% methanol, and elution of the epothilone D product was performed isocratic with 70% methanol at a flow rate of 33 mL / min. A total of 27 fractions were collected, the first fraction containing 3.8 L volume. This was followed by three 500-mL fractions and 23 250-mL fractions. Fraction 5-20 containing 4.8 g of epothilone D was obtained as the best pool (K125-179-D). Fraction 3-4 (K125-179-C) contained 1.4 g of epothilone D. Since this pool also contained high concentrations of epothilone C, it was set aside for rework (step 3b).
[590] Fourth Step Chromatography (K119-153)
[591] The epothilone D fraction, which also contains a high concentration of epothilone analog C, was re-chromatated on a C18 resin as follows. A 2.5 × 50 cm column was charged with C18 resin, washed with 1 L of 100% methanol and then equilibrated with 1 L of 55% methanol in water at a flow rate of 20 mL / min. Pressure drop was 125 psi. The starting material (K125-179-C, 1040 mL) was diluted with 260 mL of water and the loading solution contained 55% methanol in water. The resulting solution (1300 mL) was loaded onto the resin and again 250 mL of 55% methanol was passed through the column. The column was eluted first with 5 L of 65% methanol in water followed by 3 L of 70 & methanol. For 65% methanol elution, a total of 48 100-mL fractions were collected. After switching to 70% methanol, a total of 10 250-mL fractions were collected. The best epothilone D pool (K119-153-D) consisting of fractions 50-58 contained 1.0 g of the desired product.
[592] 5a step crystallization (K119-158)
[593] The starting material for this step was the combination of the chromatographic products from steps 3 and 4. First, 120 mL of ethanol was added to 7.9 g of a solid containing 5.5 g of epothilone D. With gentle mixing, the solids were completely dissolved and the solution was transferred to a 400-mL beaker placed on a stir plate in a fume hood. A 1 "stir bar was added to stir the solution rapidly. Meanwhile, about 5 100 mL of water was added slowly over minutes When small white crystals were observed to form, the solution was further stirred for 15 minutes until the solution turned to a white solid, then the beaker was removed from the stir plate and poured into aluminum foil. Covered and placed in a refrigerator (2 ° C.) for 12 hours White solids were filtered using Whatman # 50 filter paper and no further washing was done in this first harvest The solids were placed on a crystallization dish and vacuum oven (15 mbar). At 40 ° C.), then the material was removed from the oven, made into finer particles, and then further 4 hours in a vacuum oven. This crystallization process yielded 3.41 g of an off-white solid, using the Epo1 HPLC method to determine the chromatographic purity of the final product, together with the corresponding ¹ H and ¹³ C NMR data. It confirmed that it contained> 95% epothilone, and the recovery of this first harvest was 58%.
[594] 5b step crystallization (K119-167)
[595] The starting material of this step was the evaporated mother liquor from step 5a. First, 70 mL of ethanol and 30 mL of water were added to 3.4 g of a solid containing 2.1 g of epothilone. This clear solution was transferred to a beaker and 1 g of deionized charcoal was added thereto. The mixture was stirred on a fixed medium for 10 minutes and then filtered using Whatman # 50 filter paper. The charcoal was washed using two 10-mL ethanol aliquots and filtered again. The combined filtrates were dried using rotovap and the solid was resuspended in 50 mL of ethanol. The resulting solution was placed in a 250 mL beaker and 50 mL of water was slowly added while stirring well. Small amounts of seed crystals (1 mg) were added to this mixture to promote crystal formation. After stirring for a few minutes, an additional white solid was observed to form. The nitrogen stream was gently flowed around the mixture while stirring was continued. After 15 minutes, the beaker was placed in a refrigerator at 2 ° C. for 36 hours. The mixture was filtered using Whatman # 50 filter paper to capture the crystals, and then the solids were washed with 7 ml of additional 50:50 ethanol: water. The crystals were dried for 4 hours in a vacuum oven. By this crystallization step, 1.46 g of white crystals were obtained, which contained> 95% epothilone D.
[596] Fermentation carried out 1117001K
[597] First Step XAD Dissolution (K125-182)
[598] 17 liters of XAD-16 resin were filtered out of the fermentation broth 17 liters (17 L) from the fermentation broth using a Mainstream filtration unit with a 150 μm capture basket. The captured XAD resin was charged to an Amicon VA250 column and washed with 58 L (3.4 column volume) of 1.0 L / min of water. The epothilone D product was eluted from the resin with 170 L of 80% methanol in water. During water washing and during the first column volume eluting, the counter-pressure of the column was steadily raised to about 3 bar or more to achieve a final flow rate of 300 mL / min. Thus, the XAD resin was removed from the column and refilled in another Amicon VA250 column. After exchange, the back pressure decreased to less than 1 bar and the flow rate was maintained at 1.0 L / min. A single 170-L fraction was collected in a 600-L stainless steel tank. Based on HPLC analysis, the product pool of the first step was found to contain 8.4 g of epothilone D.
[599] Second Stage Solid Phase Extraction (K145-150)
[600] 57 liters (57 L) of water were added to the product pool (170 L) of the first step to dilute the loading solvent with 60% methanol in water. The resulting suspension (227L) was stirred with an overhead lighting mixer and loaded onto an Amicon VA180 column filled with 6.5-L of HP20SS resin, previously equilibrated with 5 column volumes of 60% methanol. The loading flow rate was 1 L / min. After loading, the column was washed with 16 L of 60% methanol and eluted with 84 L of 75% methanol at a flow rate of 300 mL / min. Seven fractions were obtained in volumes of 18L, 6L, 6L, 6L, 36L, 6L, and 6L, respectively. Fractions 4 and 5, together with a total of 8.8 g of epothilone D, were collected together.
[601] Third Step Chromatography (K145-160)
[602] The product pool of the second stage was evaporated to oil using two 20-L rotovaps. During evaporation, 10 L of ethanol was added to minimize bubbling. The dry material was resuspended in 2.8 L of methanol and diluted with 3.4 L of water to give 6.2 L of 45% methanol solution. The resulting solution was pumped onto a 1-L C18 chromatography column (55 × 4.8 cm) previously equilibrated with 5 column volumes of 45% methanol. The loading flow rate averaged 100 mL / min. The loaded column was washed with 1 liter of 60% methanol and the epothilone D product was eluted from the resin using a step gradient at a flow rate of 100 mL. The column was eluted with 5 L of 55% methanol, 11.5 L of 60% methanol, and 13.5 L of 65% methanol. A total of ten 500-mL fractions were obtained during elution with 55% methanol. After switching to 60% methanol, a total of 23 500-mL fractions were collected. Finally, upon eluting 65% methanol, eleven 500-mL fractions were collected, followed by eight 1-L fractions. The best epothilone D pool (K145-160-D), consisting of fractions 28-50, contained 8.3 g of the desired product. Fractions 26-27 (K145-160-C) contaminated with 0.4 g of epothilone C contained 0.2 g of epothilone D. All 25 of these fractions were combined.
[603] In order to dilute the product pool with 40% methanol in water, 9.5 L of water was added to 15.8 L of loading solution. The resulting solution (25.3 L) was pumped onto a 700-mL C-18 chromatography column (9 × 10 cm) previously equilibrated with 40% methanol 4 column volumes. The loading flow rate averaged 360 mL / min. The loaded column was washed with 1 liter of 40% methanol and the epothilone D product was eluted from the resin using 3.75 L of 100% ethanol. Eluate was evaporated to dryness using rotovap. This solid was resuspended in 100 mL of acetone and the undissolved material was filtered out of solution using Whatman # 2 filter paper. The filtered particles were washed with additional 115 mL of acetone and filtered once more. Following acetone extraction, 2 g of decolorized charcoal was added to the combined filtrates. The mixture was stirred on fixed medium for 1 hour and filtered using Whatman # 50 filter paper. The charcoal was washed with 180 mL of ethanol and filtered again. The filtrates were collected together and treated with rotovap until dry.
[604] Fourth Step Chromatography (K119-174)
[605] The dry material from the third step was resuspended in 5.0 L of 50% methanol in water and loaded onto a 1-L C18 chromatography column (55 × 4.8 cm) previously equilibrated with 50% methanol 3 column volumes. The loading flow rate averaged 80 mL / min. The column was subsequently washed with 1 liter of 50% methanol and the epothilone D product was elutically eluted from the resin with 70% methanol at the same flow rate. A total of 48 fractions were collected, the first 47 containing 240 mL and the last containing 1 L. Fractions 25-48 containing 7.4 g of epothilone D were taken as the best pool (K119-174-D). The pool also contained high concentrations of Epothilone C, which was set aside for rework.
[606] 5th Stage Crystallization (K119-177)
[607] To carry out solvent exchange prior to the recrystallization step, dilute the loading solution with 40% methanol in water by adding 3.9 L of water to 6.4 L of the best epothilone D pool (K119-174-D) from the fifth step. It was. The resulting solution was loaded onto a 200-mL C18 chromatography column (2.5 × 10 cm) previously equilibrated with 3 column volumes of 40% methanol. The Rodney column was washed with 200 mL of 40% methanol and the epothilone D product was eluted from the resin with 1 L of 100% ethanol. The eluate was evaporated to dryness using rotovap and the solid was resuspended in 150 mL of 100% ethanol. This clear solution was transferred to a beaker and slowly added 175 mL of water with good stirring. A small amount of (1 mg) seed crystals was also added to this solution to promote crystal formation. If small white crystal formation was observed, the solution was further stirred for 15 minutes until it began to thicken into a white solid. The beaker was then removed from the stir plate, covered with aluminum foil and placed in a refrigerator (2 ° C.) for 12 hours. The white solid was filtered using Whatman # 50 filter paper and no further cleaning was performed on this first crop. This solid was placed on a crystallization dish and dried for 6 hours in a vacuum oven (40 ° C. at 15 mbar). This crystallization step yielded 6.2 g of a white solid, which contained> 95% of epothilone D. The recovery rate of this first harvest stage was 74%.
[608] result
[609] The recovery of epothilone D in 1031001K was about 4.8 g of crystalline material having a purity of about 97.5 to 98.8%. The recovery of epothilone D when running 1117001K was 6.2 g of crystalline material having a purity of about 97.7%. The impurity profile of this run is shown in Table 12.
[610] 1031001K PerformstepproductEpo CEpo 490Epo D2SPE234743-4Chrom total0.70.790.65acrystallization1.01.097.55bcrystallization0.70.598.8 Perform 1117001KstepproductEpo CEpo 490Epo D2SPE182603C18 Chrom5.21.681.44C18 Chrom1.61.896.65crystallization0.81.497.7
[611] "Epo 490" is a catcher epothilone D to a novel epothilone compound of the invention, the 10,11-formaldehyde produced by mikso Rhodococcus host cell.
[612] [382] This purification method was developed as part of an effort to make a large-scale modification to the epothilone D purification method to meet the availability of methyl oleate in the fermentation medium. Elution of the epothilone D product from the XAD resin was carried out in a straightforward manner. Instead of using 100% methanol, 10 column volumes of 80% methanol were used to elute the product from the beads in the column. During the XAD elution step, it was recognized that the presence of lysed cells in the fermentation broth could cause condensation of the purification column. Harvesting of the 103100-1K fermentation run was performed before significant cell lysis occurred, and for 111700-1K fermentation, harvesting was performed only after significant cell lysis occurred. However, high back pressures and low flow rates were observed only during the latter fermentation run during the elution step. Thus, in this run, the lysed cells will aggregate with each other and later cling to the column filter.
[613] These purification steps show that epothilone D is stable in 80% methanol for at least one day at room temperature. Based on HPLC analysis, no degradation of the product was detected under these conditions. This finding allowed the 170-L product pool from the XAD elution step to be stored overnight without the need to refrigerate in a 600-L stainless steel tank. To further refine this method, a solvent-exchange column was used, which saves much time and requires less labor than using rotovap to concentrate the volume of product pool. Thus, large scale rotovap processes can be replaced by solvent-exchange steps.
[614] [384] Although during the XAD elution step a significant amount of oil remained bound to the resin, there was still a sizeable amount in the eluate. Even after HP20SS solid phase extraction, the oil droplets were clearly visible in the product pool, causing problems during C18 chromatography. For optimal chromatography performance, the concentration of epothilone D in the loading solution should be kept below 2 g / L. At higher concentrations, the starting material tends to drain oil on the column.
[615] [385] When the injected material contained more than 3% of epothilone C or Epo 490, it did not crystallize. This occurred during the purification of 1117001K. The first chromatography step produced a product containing 5% epothilone C. Despite numerous attempts, this material did not crystallize. However, reducing this material to 1% via a second chromatography step resulted in an injection material which crystallized easily.
[616] Example 4
[617] Using nonfunctional epoK gene MyxocaucusPreparation of the strain
[618] [386] Strain K111-40-1 was prepared from strain K111-32.25 by insertion inactivation of the epoK gene. To make the epoK mutant, a kanamycin resistance cassette was inserted into the epoK gene. This was done by separating the 4879 bp fragment from pKOS35-79.85 containing epoK and conjugating it to the Not I site of pBluescriptSKII +. This plasmid, pKOS35-83.5, was cleaved with Sca I and 1.5 kb Eco RI- Bam containing the kanamycin resistance gene from pBJ180-2 with DNA blunt-ended using the Klenow fragment of DNA polymerase I The plasmid pKOS90-55 was made by conjugating the 7.4 kb fragment using the HI fragment. Finally, ˜400 bp RP4 oriT fragment from pBJ183 was conjugated into the Xba I and Eco RI sites to make pKOS90-63. Cutting this plasmid with DraI, and by selecting the electrophoresis was then transformed into the body mikso Lactococcus glass tooth strain K111-32.25 provided a Rhodococcus mikso glass tooth strain K111-40.1. This strain K111-40.1 was prepared according to the Budapest Treaty under American Type Culture Collection, 10801 University Blvd. Manassas, VA, 20110-2209 USA, dated November 21, 2000, was assigned accession number PTA-2712.
[619] [387] To make the epoK mutant without markers, pKOS35-83.5 was digested with ScaI and the 2.9 kb and 4.3 kb fragments were conjugated to each other. This plasmid, pKOS90-101, had an in-frame deletion in epoK . Next, pKOS99-105 was made by conjugating NdeI and 3 kb Bam HI fragments from KG2 into the Dra I site of pKOS90-101 with DNA with blunt ends and with kanamycin resistance and galK gene by Klenow fragment of polymerase I. . This plasmid was electrophoresed into K111-32.25 to select kanamycin resistant electrophores. To replace the wild type copy of epoK by deletion, a second recombinant was selected on the galactose plate. These galactose resistant colonies were tested for the productivity of epothilones C and D, and the production strains were named K111-72.4.4, and in accordance with the Budapest Treaty, the American Type Culture Collection, 10801 University Blvd. Manassas, VA, 20110-2209 USA, dated November 21, 2000, was assigned accession number PTA-2713.
[620] Example 5
[621] matBCAddition of
[622] The matBC gene encodes malonyl Co-A synthetase and dicarboxylate carrier protein, respectively. An and Kim 1998, Eur. J. Biochem. See 257 : 395-402. These two proteins are responsible for the conversion of foreign malonates into malonyl-CoA in cells. The products of these two genes can deliver dicarboxylic acids to convert them into CoA derivatives (PCT Patent Application No. US00 / 28573, referenced herein). These two genes can be inserted into the chromosome of Myxococcus xanthus to increase polyketide productivity by increasing cell concentrations of malonyl-CoA and methylmalonyl-CoA. This cutting pMATOP-2 with Bgl II and Spe I and this tetracycline resistance gene give the att site and a Mx8, containing mikso Lactococcus glass tooth pilA promoter to drive the expression of mtaBC, pKOS35-82.1 the BglII and SpeI sites Spliced into. This plasmid can be electrophoresed into Myxococcus xanthus . Since the pilA promoter is highly transcribed, it may be necessary to insert a weaker promoter if too much MatB and MatC affect cell growth. Another promoter includes a promoter of kanamycin resistance conferring genes.
[623] Example 6
[624] KS in the loading module ^YMutation of
[625] [389] A proposed mechanism with respect to initiation of epothilone biosynthesis is that the malonate binds to the ACP of the loading domain and is subsequently decarboxylated by the loading KS domain. The loading KS domain contains tyrosine at the active site cysteine (KS ^Y ), which prevents condensation. However, it is still believed that the decarboxylation reaction is carried out. Experiments using fatty acid synthase in rats showed that KS domains containing glutamine in the active site cysteine (KS ^Q ) increased decarboxylation by a second order, while serine, alanine, asparagine, glycine or Changing to threonine did not result in any increase compared to wild type. Thus, changing KS ^Y to KS ^Q can result in an increase in epothilone priming resulting in increased epothilone production. To make changes in strain K111-32.25, plasmid pKOS39-148 was prepared to have ˜850 bp of the epothilone KS loading module coding sequence. Site directed mutations resulted in KS ^Q mutations in this plasmid. To perform gene substitution at K111-32.25, the kanamycin resistance and galK gene from KG2 were inserted at the Dra I position of pKOS39-148 to make plasmids pKOS111-56.2A and pKOS111-56.2B. These plasmids have different orientations in the kanamycin-galK cassette. These plasmids were electrophoresed into K111-32.25 to select kanamycin resistant colonies to make K111-63 strain. To replace wild type loading module KS, K111-63 was plated on CYE galactose plates and screened for the presence of KS ^Q by PCR and sequencing.
[626] Example 7
[627] mtaAAddition of
[628] [390] To increase phosphopanthethenyl transferase (PPTase) protein concentration, PPTase from Stigmatella aurantiaca strain DW4 can be added to K111-32.25. This was done by PCR amplification of mtaA from DW4 chromosomal DNA using primers 111-44.1 (AAAAGCTTCGGGGCACCTCCTGGCTGTCGGC) (SEQ ID NO: 4) and 111-44.4 (GGTTAATTAATCACCCTCCTCCCACCCCGGGCAT) (SEQ ID NO: 5). Silakowski et al., 1999, J. Biol. Chem. 274 (52): 37391-37399. ~ 800 bp was cut with NcoI and the fragment bonded to the pUHE24-2B cut with Pst I, using a DNA polymerase Klenow fragment of cyclase I made the blunt ends was digested with the following, Nco I. This plasmid was named pKOS111-54. The mtaA gene was delivered to pKOS35-82.1 containing the Mx8 att site, which is a tetracycline resistance conferring gene, and a myxococcus xanthus pilA promoter for promoting expression of mtaA . Introducing a plasmid in Rhodococcus mikso glass was incorporated into the tooth Mx8 phage attachment site.
[629] Example 8
[630] Preparation of Promoter Replacement Plasmids
[631] [391] In order to increase the level of epothilone production and to describe the various promoters that can be used to express PKS genes in host cells of the present invention, a series of vectors and host cells were made to produce the Soranium cellulose epothilone PKS gene promoter. Other suitable captive motors as described in the Examples.
[632] A. Preparation of plasmids with downstream flanking bands
[633] Cosmid pKOS35-70.8A3 was digested with Nsi I and Avr II. 9.5 kb fragment was joined to the one section pSL1190 digested with pSt groups I and Avr II made pKOS990-13. Plasmid pKOS90-13 is ˜12.9 kb. Plasmid pKOS90-13 was digested with Eco RI / Avr II. PKOS90-64 (˜8.1 kb) was made by conjugation of the 5.1 kb fragment with pBluescript digested with Eco RI / Avr II. This plasmid contains the downstream flanking zone of the promoter (some sequences upstream of the initiation codon and epoA ). The EcoRI position is ˜220 bp upstream from the epoA gene initiation codon. The Avr II site is 5100 bp downstream from the Eco RI site.
[634] B. Cloning of Upstream Flanking Bands
[635] Primers 90-66.1 and 90-67 (as described below) were cloned upstream flanking bands. Primers 90-67 are located at the 5 'end of the PCR fragment and 90-66.1 are at the 3' end of the PCR fragment. This fragment ends at 2481 bp before the start codon of the epoA gene. This ˜2.2 kb fragment was digested with Hind III. Klenow polymerase was added to blunt the Hind III site. This fragment was joined to the Hinc II located in pNEB193. Clones with appropriate orientation, with EcoR I position at the downstream end of the insert and Hind III position at the upstream end of the insert, were selected and named pKOS90-90.
[636]
[637]
[638] C. Preparation of the Final Plasmid
[639] [394] Plasmid pKOS90-90EcoRIHindIIICut to This 2.2 kb fragmentEcoRI / HindIIIPKOS90-91 (10.3 kb) was made by conjugation with pKOS90-64 digested with. Plasmid pKOS90-91 contains the upstream flanking band of the promoter in the pBluscript followed by the downstream flanking band. Between two flanking bands, for cloning the desired promoterPacIThere is a location.galK / kan ^r Insert the cassetteMyxocaucus XanthusWas recombined. Plasmid pKOS90-91DraICut into pieces.ampIn genesDraIOnce, twice in a vector (ampNear the gene). Plasmid KG-2BamHI / NdeIAnd fragments were blunted by addition of Klenow polymerase. This 3kb fragment (galK / kan ^r Gene) was conjugated with the 9.8 kb DraI fragment of pKOS90-91 to make pKOS90-102 (12.8 kb).
[640] D. Preparation of Plasmids Using Another Reader
[641] [395] The native leader zone of the epothilone PKS gene can replace a leader with different ribosomal binding sites. Plasmid pKOS39-136 (described in US Patent Application No. 09 / 443,501, Nov. 19, 1999) was digested with PacI / Asc I. A 3 kb fragment containing the leader sequence and a portion of epoA was isolated and conjugated with the 9.6 kb PacI / AscI fragment of pKOS90-102 to make pKOS90-106 (˜12.7 kb).
[642] E. Preparation of Promoter Substituted Plasmids
[643] I. MTA (myxothiazole) promoter
[644] [396] primer using a 111 to 44.3 111 to 44.5 and stigmasterol telra brother were amplified PCR is a thiazol-promoter from mikso thiazol car of chromosomal DNA (strain DW4) (described below). By cloning the ~ 554 bp Hinc II in the position of pNEB193 made pKOS90-107. Plasmid pKOS90-107 was digested with Pst I and Xba I to be Klenow filled. The 560 bp band was cloned into pKOS90-102 and pKOS90-106 was cleaved with Pac I and Klenow charged (PacI cleavage only once in pKOS90-102 and pKOS90-106). Plasmids were screened for correct orientation. The MTA promoter / pKOS90-102 plasmid was named pKOS90-114 (13.36 kb) and the MTA promoter / pKOS90-106 plasmid was named pKOS90-113 (13.26 kb).
[645]
[646] [397] These plasmids were electrophoresed to myxococcal host cells containing the epothilone PKS gene to identify kanamycin resistant transformants to identify single crossover recombinants. These transformants were selected for galactose resistance to identify double crossover recombinants and screened by Southern analysis and PCR to identify those with the desired recombinants. Desired recombinants were cultured to test for epothilone production.
[647] II. TA promoter
[648] [398] The putative promoters for TA with taA , encoding putative transcriptional anti-terminators, were designated primers 111-44.8 (AAAGATCTCTCCCGATGCGGGAAGGC) (SEQ ID NO: 10) and 111-44.9 (GGGGATCCAATGGAAGGGGATGTCCGCGGAA). (SEQ ID NO: 11) was used to amplify from strain TA. An about 1.1 kb fragment was digested with Bam HI and Bgl II and conjugated to pNEB193 digested with Bam HI. This plasmid was named pKOS111-56.1. Plasmid pKOS111-56.1 was digested with EcoRI and HindIII and Klenow filled. The ˜1.1 kb band was cloned into pKOS90-102 and pKOS90-106 was cleaved with Pac I and then Klenow charged ( PacI cleavage only once in pKOS90-102 and pKOS90-106). Plasmids were screened for correct orientation. The TA promoter / 90-102 plasmid was named pKOS90-115 (13.9 kb) and the TA promoter / pKOS90-106 plasmid was named pKOS90-111 (13.8 kb).
[649] [0103] These plasmids were electrophoresed to myxococcal host cells containing the epothilone PKS gene, and kanamycin resistant transformants were selected to identify single crossover recombinants. These transfectants were selected for galactose resistance to identify double crossover recombinants. Screened by Southern analysis and PCR to identify whether the desired recombination occurred. These desired recombinants were cultured to investigate the epothilone productivity.
[650] III. pilAPromoter
[651] [400] Plasmid pKOS35-71B was digested with Eco RI and Klenow filled. 800 bp fragment was cloned into pKOS90-12 and pKOS90-106 was digested with Pac I and Klenow charged. Plasmids were screened for correct orientation. The PilA promoter / pKOS90-102 was named pKOS90-120 (13.6 kb) and the pilA promoter / pKOS90-106 plasmid was named pKOS90-121 (13.5 kb).
[652] The plasmids were electrophoresed with myxococcal host cells containing the epothilone PKS gene to select kanamycin resistant transformants to identify single crossover recombinants. These transformants were screened for galactose resistance to identify double crossover recombinants, which were screened by Southern analysis and PCR to examine whether these desired recombinations occurred. Desired recombinants were cultured to investigate epothilone productivity.
[653] IV. kan promoter
[654] Plasmid pBJ108-2 was digested with BamHI / BglII to be Klenow charged. 350 bp fragment was cloned into pKOS90-102 and pKOS90-106 was cleaved with PacI and Klenow filled. Plasmids were screened for correct orientation. The kan promoter / pKOS90-102 plasmid was named pKOS90-126 (13.15 kb) and the kan promoter pKOS / 90-106 plasmid was named pKOS90-122 (13/05 kb).
[655] [403] These plasmids were electrophoresed to myxococcal host cells containing the epothilone PKS gene and kanamycin resistant transformants were selected to identify single crossover recombinants. This transformant was screened for galactose resistance to identify double crossover recombinants, which were screened by Southern analysis and PCR to identify the desired recombinants. Desired recombinants were cultured and tested for epothilone production.
[656] V. So ce90 promoter
[657] The So ce90 promoter was amplified from So ce90 chromosomal DNA using primers 111-44.6 and 111-44.7 (as follows). ~ 900 bp band was cut to a PacI cloned into the PacI cut into pNEB193 made pKOS90-125. Plasmid pKOS990-125 was digested with PacI . The 924 bp band was cloned into pKOS990-102 and pKOS90-106 was cleaved with PacI . Plasmids were screened for correct orientation. The Soce90 promoter / pKOS90-102 plasmid was named pKOS90-127 (13.6 kb) and the Soce90 promoter / pKOS90-106 plasmid was named pKOS990-128 (13.7 kb).
[658] These plasmids were electrophoresed to myxococcal host cells containing the epothilone PKS gene and kanamycin resistant transformants were selected to identify single crossover recombinants. This transformant was screened for galactose resistance to identify double crossover recombinants, which were screened by Southern analysis and PCR to identify the desired recombinants. Desired recombinants were cultured and tested for epothilone production.
[659]
[660] Example 9
[661] Preparation of KS2 Knockout Strains
[662] This example describes a method for preparing epothilone PKS in which the KS domain of extender module 2 is inactivated by a mutation that changes an active site, a cysteine codon, to an alanine codon. The obtained precursor PKS can be provided with a synthetic precursor (as described in the following examples) to make the epothilone derivatives of the present invention.
[663] [408] The downstream flanking band of the epothilone PKS gene was determined by primers 90-103 (5'-AAAAAATGCATCTACCTCGCTCGTGGCGGTT-3 ') (SEQ ID NO: 14) and 90-107.1 (5'-CCCCCTCTAGA ATAGGTCGGCAGCGGTACCCG) from plasmid pKOS35-78.2. -3 ') (SEQ ID NO: 15) using PCR amplification. ˜2 kb PCR product was cleaved with NsiI / Xba I and conjugated to pSL11990 with Nsi I and Spe I to make pKOS90-123 (˜5.4 kb). ˜2 kb PCR fragment was digested from primers 90-105 (5′-TTTTTATGCATGCGGCAGTTTGAACGG-AGATGCT-3 ′) (SEQ ID NO: 16) and primers 90-106 (5′-CCCCCGAATTCTCCCGGAAGGCAC ACGGAGAC-3 ′) from pKOS35-78.2 NO: 17). DNA was cleaved with Nsi I and conjugated to pKOS90-123 cleaved with NsiI / EcoRV to make pKOS90-130 (˜7.5 kb). When this plasmid was cleaved with Nsi I, the DNA end was blunt-ended with a Koenow fragment of DNA polymerase I, and rejoined to make plasmid pKOS90-131. To clone the galK / kan ^r cassette into this plasmid, plasmid KG-2 was digested with BamHI / Nde and blunt-ended using Klenow fragment of DNA polymerase I. The 3 kb fragment was cloned into the DraI position of pKOS90-131 ( DraI cleaved three times in the vector) to create plasmid pKOS90-132 (10.5 kb). The Nsi I position is used for the purpose of performing KOS2 knockout by changing cysteine to alanine as desired. If pKOS90-130 was cleaved with NsiI , blunt ended with Klenow fragments from DNA polymerase I, reconjugated and substituted the cysteine codons with alanine codons. The obtained plasmid can be introduced into the myxococcus xanthus strain of the present invention according to the above protocol to make a desired strain.
[664] [408] Myxococcus xanthus strain K90-132.1.1.2 was prepared by this method (using K111-32.25, producers of Epothilones A, B, C and D) and in accordance with the Budapest Treaty, American Type Culture Collection, 10801. University Blvd. Manassas, VA, 20110-2209 USA, dated November 21, 2000, was assigned accession number PTA-2715. To demonstrate that the PKS produced by this strain can synthesize epothilones when provided with the appropriate "dketide" initiation unit, strain K90-132.1.1.2 was used in three 30 minute portions of 50 mL of CTS plus 90% XAD. Incubated at &lt; RTI ID = 0.0 &gt; C, &lt; / RTI &gt;
[665]
[666] This strain was incubated for 5 more days, and XAD was collected to extract 10% methanol of epothilone. The extract was dried and resuspended in 0.2 ml of acetonitrile and LC / MS analysis of 0.05 mL samples showed the presence of epothilones B and D as expected. As discussed in the following examples, this system can be used to produce various epothilone analogs.
[667] Example 10
[668] Modified Epothilones from Chemical Biosynthesis
[669] This example illustrates a series of thioesters for the production of epothilone derivatives via chemical biosynthesis. The DNA sequence of the biosynthetic gene cluster for producing epothilones from Sorangium cellulose indicates that priming of PKS is associated with a mixture of polyketide and amino acid components. Priming is associated with malonyl CoA loading of the PKS-like site of the loading module followed by decarboxylation and cysteine loading of extender module 1 NPRS, followed by condensation for enzyme-linked N-acetylcysteine formation. Following cyclization for thiazolin formation, oxidation occurs to yield 2-methylthiazole-4-carboxylate bound enzyme, the product of the loading module and NPRS. Subsequent condensation with methylmalonyl CoA by ketocinase of module 2 provides a diketide equivalent as shown in Scheme 6 below.
[670]
[671] [410] The present invention relates to PCT Publication Nos. Biosynthesis and reagents for producing epothilone derivatives are provided in a similar way to making 6-dEB and erythromycin described in 99/03986 and 97/02358. Two kinds of infusion substrates are provided: analogs of NRPS products and analogs of diketide equivalents. NRPS product analogs are used with PKS enzyme with mutated NRPS-like domains, and diketide equivalents are used with PKS enzyme with KS domains mutated in Module 2 (as described in Example 9). In the structures of Schemes 7 and 8 described below, R, R ₁ and R ₂ may be independently selected from methyl, ethyl, lower alkyl (C _1-6 ) and substituted lower alkyl.
[672] Scheme 7 below illustrates a loading module analog.
[673]
[674] Loading module analogs are prepared by activating the corresponding carboxylic acid and treating it with N-acetylcysteamine. Activation methods include the formation of acid chlorides, the formation of mixed anhydrides or reaction with condensation reagents such as carbodiimides.
[675] Scheme 8 below illustrates a diketide equivalent.
[676]
[677] Diketide equivalents were prepared in a three step process. First, the corresponding aldehyde is treated with Wittig reagent or equivalent to form a substituted acrylic ester. This ester is saponified to an acid, which is activated and treated with N-acetylcysteamine.
[678] [413] An exemplary scheme for preparing the loading module product analogues and diketide equivalents is as follows. Additional compounds suitable for making diketide equivalents are shown in FIG. 1 as carboxylic acids (or aldehydes which can be converted to carboxylic acids) which are converted to N-aciessteamides to be supplied to the host cells of the invention.
[679] A. Thiophene-3-carboxylate N-acedsteamine thioester
[680] Drying under Inert Atmosphere A solution of thiophene-3-carboxylic acid (128 mg) in 2 mL of tetrahydrofuran was treated with triethylamine (0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour, N-acetylcysteamine (0.25 mL) was added and the reaction proceeded for 12 hours. The mixture was poured into water and extracted three times with eastern blood ethyl acetate. The organic extracts were combined, washed sequentially with water, 1N HCl, saturated CuSO ₄ and brine, dried over MgSo ₄ , filtered and concentrated in vacuo. Chromatography on SiO ₂ with ether followed by ethyl acetate gave a pure product which crystallized upon standing.
[681] B. Furan-3-carboxylate N-acetylcysteamine thioester
[682] Under an inert atmosphere, a solution of furan-3-carboxylic acid (112 mg) in 2 mL of dry tetrahydrofuran was treated with triethylamine (0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour, N-acetylcysteamine (0.25 mL) was added and the reaction proceeded for 12 hours. The mixture was poured into water and extracted three times with eastern blood ethyl acetate. The organic extracts were combined, washed sequentially with water, 1N HCl, saturated CuSO ₄ and brine, dried over MgSo ₄ , filtered and concentrated in vacuo. Chromatography on SiO ₂ with ether followed by ethyl acetate gave a pure product which crystallized upon standing.
[683] C. Pyrrole-2-carboxylate N-acetylcysteamine thioester
[684] In an inert atmosphere, a solution of pyrrole-2-carboxylic acid (112 mg) in 2 mL of dry tetrahydrofuran was treated with triethylamine (0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour, N-acetylcysteamine (0.25 mL) was added and the reaction proceeded for 12 hours. The mixture was poured into water and extracted three times with eastern blood ethyl acetate. The organic extracts were combined, washed sequentially with water, 1N HCl, saturated CuSO ₄ and brine, dried over MgSo ₄ , filtered and concentrated in vacuo. Chromatography on SiO ₂ with ether followed by ethyl acetate gave a pure product which crystallized upon standing.
[685] D. 2-Methyl-3- (3-thienyl) acrylate N-acetylcysteamine thioester
[686] (1) Ethyl 2-methyl-3- (3-thienyl) acrylate : thiophene-3-carboxaldehyde (1.12 g) and (carbethoxye) in dry tetrahydrofuran (20 mL). The mixture of tilidene) triphenylphosphorane (4.3 g) was heated to reflux for 16 hours. The mixture was cooled to ambient temperature and concentrated in vacuo to dryness. The solid residue was suspended in 1: 1 ether / hexanes and filtered to remove triphenylphosphine oxide. The filtrate was filtered through a pad of SiO ₂ with 1: 1 ether / hexanes to give the product (1.78 g, 91%) as a pale yellow oil.
[687] (2) 2-Methyl-3- (3-thienyl) acrylic acid : The ester from step (1) was dissolved in a mixture of methanol (5 mL) and 8N KOH (5 mL) and heated to reflux for 30 minutes. The mixture was cooled to ambient temperature, diluted with water and washed twice with ether. The aqueous phase was acidified with 1N HCl and then extracted three times with an equal volume of ether. The combined organic extracts were dried over MgSO ₄ , filtered and concentrated to dryness in vacuo. Crystallization from 2: 1 hexanes / ether gave a colorless needles.
[688] [419] (3) 2-Methyl-3- (3-thienyl) acrylate N-acetylcysteamine thioester : 2-methyl-3- (3-thienyl) in 2 mL of tetrahydrofuran dried under an inert atmosphere. A solution of acrylic acid (168 mg) was treated with triethylamine (0.56 mL) and diphenylphosphoryl azide (0.45 mL). After 15 minutes, N-acetylcysteamine (0.15 mL) was added and the reaction proceeded for 4 hours. The mixture was poured into water and extracted three times with equal volume of ethyl acetate. The organic extracts were combined, washed sequentially with water, 1N HCl, saturated CuSO ₄ and brine, dried over MgSo ₄ , filtered and concentrated in vacuo. Chromatography on SiO ₂ with ethyl acetate gave the pure product which crystallized upon standing.
[689] The compound is supplied to a culture of a host cell containing the recombinant epothilone PKS of the present invention, wherein the NRPS or KS domain of extender module 2 is inactivated by a mutation to produce the corresponding epothilone derivative of the present invention. .
[690] Example 11
[691] Production of Epothilone Analogs
[692] A. Production of 13-keto-epothilone analogs
[693] [421] Inactivation of the KR domain in extender module 4 of epothilone PKS yields the hybrid PKS of the invention useful for 13-keno epothilone production. The extender module 4 KR domain was modified by replacing wild type genes with various deletion versions as described below. First, fragments were amplified using plasmid pKOS39-118B (subclone of the epoD gene from cosmid pKOS35-70.4) as a template. Oligonucleotide primers forming the deletion left were TL3 and TL4 as shown below:
[694] TL3: 5'-ATGAATTCATGATGGCCCGAGCAGCG (SEQ ID NO: 18) and
[695] TL4: 5'-ATCTGCAGCCAGTACCGCTGCCGCTGCCA (SEQ ID NO: 19) ..
[696] Oligonucleotide primers forming the deletion right side were TL5 and TL6 as follows:
[697] TL5: 5'-GCTCTAGAACCCGGAACTGGCGTGGCCTGT (SEQ ID NO: 20) and
[698] TL6: 5-GCAGATCTACCGCGTGAGGACACGGCCTT (SEQ ID NO: 21).
[699] PCR fragments were cloned into the vector Litmus 39 and sequenced to confirm that the desired fragments were obtained. The clones containing the TL3 / TL4 fragment were then digested with restriction enzymes Pst I and Bam HI to separate ˜4.6 kb fragments. 2.0 kb PCR fragments obtained using primers TL5 / TL6 were treated with restriction enzymes Bgl II and Xba I followed by (i) "short" KR linkers TL23 and TL24 (annealed together to form double chain linkers with single-chain overhangs). To form pKOS122-29; Or (ii) binding to a "long" (epoDH3 *) linker, obtained by PCR with primer TL33 + TL34 followed by restriction enzymes NsiI and SpiI treatment to obtain plasmid pKOS122-30. The sequences of these oligonucleotide linkers and primers are as follows:
[700]
[701] [422] Plasmids containing the desired substitutions were confirmed by sequencing and then digested with restriction enzyme DraI. Large fragments of each clone were then conjugated to kanamycin resistance and galK gene (KG or kan-gal ) cassettes to create a plasmid for delivery. By using this transfer plasmid it was transformed by the epothilone B producer Rhodococcus mikso glass tooth K111-32.25 electrophoresis. Transformants were screened to screen kanamycin-sensitive, galactose-resistant survivors to identify clones from which the KG gene was removed. KG clearance was confirmed and the desired gene replacement by recombinant strain was performed by PCR. Recombinant strains were fermented in flasks with 50 mL of CTS medium and 2% XAD-16 for 5 days to elute the epothilone analog with XAD and 10 mL methanol. Structure determination was based on LC / MS spectra and NMR. One such strain is named K122-56 and in accordance with the Budapest Treaty, the American Type Culture Collection, 10801 University Blvd. Manassas, VA, 20110-2209 USA, dated November 21, 2000, was assigned accession number PTA-2714. K122-56 strain (derived from plasmid VI122-29) produces 13-keto-11,12-dehydro-epothilone D having the following structure as the main product:
[702]
[703] [423] The K122-56 strain also produces 13-keto-epothilones C and D, each having the following structure, as side products:
[704]
[705] Similar results were obtained with strain K122-30 derived from plasmid pKOS122-30. These compounds and strains and PKS enzymes that produce them are novel compounds, strains and PKS enzymes of the invention.
[706] [425] Other strains of the invention that produce 13-keto-11,12-dehydroepothilone include those in which the KR domain is inactivated by one or more point mutations. For example, mutation of a member tyrosine residue in the KR domain with a phenylalanine residue results in about 10% reduction in KR activity and results in 13-keto-epothilone production. Additional mutations in the KR domain may additionally or eliminate all KR activity, but may also result in decreased epothilone production.
[707] B. Production of 13-hydroxy-epothilone analogs
[708] [426] Substitution of extender modules 5KR, DH and ER domains of epothilone PKS with heterologous KR domains, such as extender module 2 of rapamycin PKS or KR module from extender module 3 of FK520 PKS, resulted in 13-hydroxy epothilone production. A useful inventive hybrid PKS is obtained. This is done in a manner similar to that described in Part A of this embodiment. Oligonucleotide primers for amplifying the desired portion of the epoD gene using plasmid pKOS39-118B as a template were as follows:
[709] TL7: 5'-GCGCTCGAGAGCGCGGGTATCGCT (SEQ ID NO: 26);
[710] TL8: 5'-GAGATGCATCCAATGGCGCTCACGCT (SEQ ID NO: 27);
[711] TL9: 5'-GCTCTAGAGCCGCGCGCCTTGGGGCGCT (SEQ ID NO: 28) and
[712] TL10: 5-GCAGATCTTGGGGCGCTGCCTGTGGAA (SEQ ID NO: 29).
[713] [427] PCR fragments produced from primers TL7 / TL8 were cloned into vector LITMUS 28, and the resulting clones were digested with restriction enzymes Nsi I and Bgl II to isolate 5.1 kb fragments and treated with restriction enzymes Bgl II and Xba I. It was conjugated to a KR cassette by conjugation with a 2.2 kb PCR fragment generated from TL9 / TL10. KR cassettes from FK520 PKS produced by PCR using primers TL31 and TL32 were cleaved using restriction enzymes Xba I and Pst I. These primers are as follows:
[714]
[715] [428] The rest of the strains are prepared and proceeds in a manner analogous to that described in part A of this example except that the cup is mikso Lactococcus tooth K111-72.44 used as the receptor. The KR domain of the extender module 3 of the extender module of the FK520 PKS replaced the KR, DH and ER domains of the extender module of the epothilone PKS as K122-148, and according to the Budapest Treaty, the American Type Culture Collection, 10801 University Blvd. Manassas, VA 20110-2209 USA, dated November 21, 2000, was assigned accession number PTA-2711. Strain K122-148 produces 13-hydroxy-10,11-dehydro epothilone D as the main biological product and epothilone C derivative as a by-product and has the following structure:
[716]
[717] [429] Similar strain K122-52, in which the KR domain of extender module 2 of rapamycin PKS was used for substitution, produced the same compound. These compounds and strains and PKS enzymes producing them were the novel compounds, strains and PKS enzymes of the invention.
[718] C. Production of 9-keto-epothilone analogs
[719] [430] Inactivation of the KR domain of extender module 6 of epothilone PKS results in a novel PKS of the invention capable of producing 9-keto-epothilone. KR domains can be activated by site-specific mutations by altering one or more conserved residues. The DNA and amino acid sequences of the KR domain of extender module 6 of epothilone PKS are as follows:
[720]
[721]
[722] [431] The DNA and amino acid sequences of the mutated and inactive KR domain of extender module 6 of the novel 9-keto-epothilone PKS provided by the present invention are as follows:
[723]
[724]
[725] [432] As a receptor, except that the glass mikso Lactococcus tooth k111-72.4.4, was produced in a strain containing the mutated KR domain coding sequence by the present embodiment A description part doengeot and generally in the same way in. This strain, inactivated by the KR domain of extender module 6, was named K39-164 and under the Budapest Treaty, the American Type Culture Collection, 10801 University Blvd. Manassas, VA 20110-2209 USA, dated November 21, 2000, was assigned accession number PTA-2716. Strain K39-164 produces 9-keto-epothilone D as a main product and C derivatives as a by-product and has the following structure:
[726]
[727] These compounds and strains and PKS enzymes that produce them are novel compounds, strains and PKS enzymes of the invention.
[728] D. Production of 2-methyl-epothilone analogs
[729] [433] 2-methyl-epothilone analogs of epothilones A, B, C and D replace the coding sequence of the extender module 9 AT domain (“epoAT9”) with the coding sequence of the AT domain specific for methylmalonyl CoA. It can produce by making it. Thus, suitable substitution AT domain coding sequences are described, for example, in Extender Module 2 of FK520 PKS (“FKAT2”; PCT Publication No. 00/020601, referenced herein); Extender module 2 of Epothilone PKS ("epoAT2"); And extender module 3 of PKS encoded by the tmbA gene ("tmbAT3"; U.S. Patent No. 6,090601 and U.S. Patent Application No. 60 / 271.245, referenced February 15, 2001). Can be. Such substitutions are generally carried out as described above, and the specific epothilones produced are only dependent on which epothilones are produced by the myxococcus host from which the substitutions were induced.
[730] Thus, the epoAT9 coding sequence (nucleotides 50979 to nucleotide 52026) is either epoAT2 (nucleotides 12251 to nucleotide 13287) or FKAT2, or engineered BglII ( ATATCT ) and NsiI ( ATGCAT ) restriction enzymes in junction, with tmbAT3 coding sequences. It is substituted with recognition sequences.
[731] [435] The first PCR was used to produce ˜1.6 kb fragments from pKOS39-125 DNA used as template. This PCR fragment is sequenced by subcloning into vector LITMUS28 at the Hind III and Bgl II positions; The plasmid with the desired sequence is called P1. Oligonucleotides used in this PCR are as follows:
[732] TLII-1: 5'-ACAAGCTTGCGAAAAAGAACGCGTCT (SEQ ID NO: 36); And
[733] TLII-2: 5'-CGAGATCTGCCGGGCGAGGAAGCGGCCCTG (SEQ ID NO: 37).
[734] [436] The second PCR is used to produce ~ 1.9 kb fragments from pKOS39-125 DNA used as template. This PCR fragment is sequenced by subcloning into vector LITMUS28 at the Nsi I and Spe I positions; The plasmid with the desired sequence is called P2. Oligonucleotides used in this PCR are as follows:
[735] TLII-3B: 5'-GCATGCATGCGCCGGTCGATGGTGAG (SEQ ID NO: 38); And
[736] TLII-4: 5'-AGACTAGTCACCGGCTGGCCCACCACAAGG. (SEQ ID NO: 39)
[737] [437] Plasmid P1 was digested with restriction enzymes BglII and SpeI to separate 4.5 kb fragments and three substituted AT fragments isolated as ~ 1.9 kb NsiI-SpeI restriction enzyme fragment and NsiI-BglII restriction enzyme fragment from plasmid P2 ( Conjugation with one of FKAT2, epoAT2, tmbAT3) to give plasmids P3.1, P3.2 and P3.3. Substituted AT fragments are produced by PCR using the following oligonucleotide primers:
[738]
[739] [438] Plasmids P3.1, P3.2 and P3.3 are modified by inserting into the DraI position of the kan-gal cassette. The resulting plasmid is transformed into myxococcus xanthus host cells (ie, K111-72.4.4) producing the epothilones of the invention, and the cells are cultured and selected for double-crossover recombination as described above. . Selected colonies are screened by PCR. Colonies resulting from the desired recombination were incubated in 50 mL cultures to screen for the production of the desired compounds by LC / MS. Expected products are 2-methyl-epothilone D and 2-methyl-epothilone C whose structure is as follows.
[740]
[741] E. Production of 6-Desmethyl-Epothilone Analogues
[742] [439] 6-desmethyl-epothilone analogs of epothilones A, B, C, and D are made by replacing the coding sequence of the extender module 7 AT domain (“epoAT7”) with the coding sequence of the malonyl CoA specific AT domain. Can be. Thus, suitable substitution AT domain coding sequences are described, for example, in extender module 3 of FK520 PKS; Extender module 5 of epothilone PKS (“epoAT5”); And extender module 4 of PKS encoded by the tmbA gene. Such substitutions are generally carried out as described above, and the specific epothilones produced are only dependent on which epothilones are produced by the myxococcus host from which the substitutions were induced.
[743] [440] Thus, the epoAT7 coding sequence (nucleotides 39585 to nucleotide 40626) is either epoAT5 (nucleotides 26793 to nucleotide 27833) or FKAT3, or engineered BglII ( AGATCT ) and NsiI ( ATGCAT ) restriction enzymes in junction, with tmbAT4 coding sequences. It is substituted with recognition sequences.
[744] [441] The first PCR was used to produce ˜1.8 kb fragments from pKOS39-125 DNA used as template. This PCR fragment is sequenced by subcloning into the vector LITMUS28 at the NSi I and Spe I positions; The plasmid with the desired sequence is called P4. Oligonucleotides used in this PCR are as follows:
[745] LII-5: 5'-GGATGCATGTCGAGCCTGACGCCCGCCG (SEQ ID NO: 46); And
[746] LII-6: 5'-GCACTAGTGATGGCGATCTCGTCATCCGCCGCCAC (SEQ ID NO: 47)
[747] The second PCR is used to produce ˜2.1 kb fragment using pKOS039-118B as a template. Oligonucleotides used in this PCR are as follows:
[748] TL16: ACAGATCTCGGCGCGCTGCCGCCGGAG (SEQ ID NO: 48) and
[749] TL15: GGTCTAGACTCGAACGGCTCGCCACCGC (SEQ ID NO: 49).
[750] The PCR fragment was subcloned into LITMUS28 at the EcoRV restriction enzyme position and the plasmid having the desired sequence was identified by sequencing and named plasmid pKOS122-4. This plasmid pKOS122-4 was digested with restriction enzymes BglII and SpeI to separate 4.8 kb fragments and three substituted AT fragments isolated as ~ 1.8 kb NsiI-SpeI restriction enzyme fragment and NsiI-BglII restriction enzyme fragment from plasmid P4 ( Conjugation with one of FKAT3, epoAT5, tmbAT4) to obtain plasmids P5.1, P5.2 and P5.3. Substituted AT fragments are produced by PCR using the following oligonucleotide primers:
[751] FKAT3:
[752] TLII-11: 5'-GTATGCATCCAGTAGCGGACCCGCTCGA (SEQ ID NO: 50); And
[753] TLII-12: 5'-GCAGATCTGTGTGGCTCTTCTCCGGACA (SEQ ID NO: 51);
[754] tmbAT4:
[755] TLII-15; 5'-GCATGCATCCAGTAGCGCTGCCGCTGGAAC (SEQ ID NO: 52); And
[756] TLII-16; 5'-GGAGATCTGCGGTGCTGTTCACGGGGCA (SEQ ID NO: 53); And
[757] PCR epoAT5:
[758] TLII-19; 5'-GTAGATCTGCTTTCCTGTTCACCGGACA (SEQ ID NO: 54); And
[759] TL8 (see part of this example).
[760] Plasmids P5.1, P5.2 and P5.3 are modified by insertion at the kan-gal cassette Dra I position. The resulting plasmids were transformed into the epothilone producing myxococcus xanthus host cells (ie, k111-72.4.4) of the present invention, and the cells were cultured and selected for double-crossover recombinants as described above. Selected colonies were screened by PCR. Colonies representing the desired recombination were cultured in 50 mL cultures to screen for the production of the desired product by LC / MS. Expected compounds are 6-desmethyl-epothilone D and 6-desmethyl-epothilone C whose structure is as follows:
[761]
[762] F. Production of 10-methyl-epothilone analogs
[763] [445] The 10-methyl-epothilone analogues of epothilones A, B, C, and D are made by replacing the coding sequence of extender module 5 AT domain (“epoAT5”) with the coding sequence of methylmalonyl CoA specific AT domain. Can be. Thus, suitable substitution AT domain coding sequences are described, for example, in extender module 2 of FK520 PKS; Extender module 2 ("epoAT2") of epothilone PKS; And the gene encoding extender module 3 of PKS encoded by the tmbA gene. Such substitutions are generally carried out as described above, and the specific epothilones produced are only dependent on which epothilones are produced by the myxococcus host from which the substitutions were induced.
[764] Thus, the epoAT5 coding sequence (nucleotides 26793 to nucleotide 27833) is either epoAT2 (nucleotides 12251 to nucleotide 13287) or FKAT2, or engineered BglII ( AGATCT ) and NsiI ( ATGCAT ) restriction enzymes in junction, with tmbAT3 coding sequences. It is substituted with recognition sequences.
[765] PCR fragments prepared from primers TL11 and TL12 were cloned into vector LITMUS 28 using plasmid pKOS39-118B as a template. PCR primers used were as follows:
[766] TL11: 5'-GGATGCATCTCACCCCGCGAAGCG (SEQ ID NO: 55) and;
[767] TL12: 5'-GTACTAGTCAAGGGCGCTGCGGAGG (SEQ ID NO: 56).
[768] [447] Plasmids containing the desired inserts were identified by DNA sequencing. This plasmid was digested with restriction enzymes NsiI and XbaI and the 4.6 kb fragment isolated. 2.0 kb PCR fragment obtained from primers Tl5 and TL6 digested with restriction enzymes BglII and XbaI (described in section A of this example), and three substituted AT fragments (FKAT2) isolated as NsiI-BglII restriction enzyme fragments. , epoAT2, tmbAT3) to obtain plasmids P6.1, P6.2 and P6.3. These latter three plasmids were modified by inserting at the DraI position of the kan-gal cassette. The resulting plasmids were transformed into the epothilone producing myxococcus xanthus host cells (ie, K111-72.4.4) of the present invention, and the cells were cultured and selected for double-crossover recombinants as described above. Selected colonies were screened by PCR. Colonies representing the desired recombination were cultured in 50 mL cultures to screen for the production of the desired product by LC / MS. Expected compounds are 10-methyl-epothilone D and 10-methyl-epothilone C whose structure is as follows:
[769]
[770] G. Production of 14-Methyl-Epothilone Analogues
[771] [448] 14-methyl-epothilone analogs of epothilones A, B, C, and D are made by replacing the coding sequence of extender module 3 AT domain (“epoAT3”) with the coding sequence of methylmalonyl CoA specific AT domain. Can be. Thus, suitable substitution AT domain coding sequences are described, for example, in extender module 2 of FK520 PKS; Extender module 2 ("epoAT2") of epothilone PKS; And the gene encoding extender module 3 of PKS encoded by the tmbA gene. Such substitutions are generally carried out as described above, and the specific epothilones produced are only dependent on which epothilones are produced by the myxococcus host from which the substitutions were induced.
[772] [449] Thus, the epoAT3 coding sequence (nucleotides 17817 to nucleotide 18858) may be engineered at the junction with epoAT2 (nucleotides 12251 to nucleotide 13287) or FKAT2, or tmbAT3 coding sequence, Bgl II ( AGATCT ) and Nsi I ( ATGCAT ) It is substituted with restriction enzyme recognition sequences.
[773] [450] The first PCR was used to make ~ 1.8kb fragments from pKOS39-124 DNA used as template. This PCR fragment was cloned into vector LITMUS 28 at the Xba I and Bgl II positions and then sequenced; The plasmid with the desired sequence was named P9. Oligonucleotides used in this PCR are as follows:
[774] TLII-'7: 5'-GCAGATCTGCCGCGCGAGGAGCTCGCGAT (SEQ ID NO: 57 :) and
[775] TLII-8: 5'-CATCTAGAGCCGCTCCTGTGGAGTCAC (SEQ ID NO: 58).
[776] [451] The second PCR is used to make a ~ 1.9 kb fragment using pKOS39-124 DNA as a template. This PCR fragment was cloned into vector LITMUS 28 at NSi I and Spe I positions and then sequenced; The plasmid with the desired sequence was named P10. Oligonucleotides used in this PCR are as follows:
[777] TLII-9B: 5'-GGATGCATGCGCCGGCCGAAGGGCTCGGAG (SEQ ID NO: 59); And
[778] TLII-10: 5'-GCACTAGTGATGGCGATCGGGTCCTCTGTCGC (SEQ ID NO: 50).
[779] [452] Plasmid P9 was cleaved with restriction enzymes Bgl II and Spe I, 4.5 kb fragments were isolated, and isolated as ~ 1.9 kb NsiI-Spe I restriction enzyme fragment and NsiI-Bgl II restriction enzyme fragment from plasmid P10. Conjugation with one of three substitutional AT fragments (FKAT2, epoAT2, tmbAT3) yields plasmids P11.1, P11.2 and P11.3. These latter three plasmids were modified by inserting at the Dra I position of the kan-gal cassette. Were selected for the crossover recombinants - the resulting plasmid was transformed into double, as epothilone production mikso Lactococcus glass tooth host cell (i.e., K111-72.4.4) of the present invention, the above-mentioned cultured cells. Selected colonies were screened by PCR. Colonies representing the desired recombination were cultured in 50 mL cultures to screen for the production of the desired product by LC / MS. Expected compounds are 14-methyl-epothilone D and 14-methyl-epothilone C whose structure is as follows:
[780]
[781] H. Production of 10,11-dehydro-epothilone analogs
[782] [453] In one embodiment, the present invention provides novel epothilones, 10,11-dehydro-epothilone D, and recombinant host cells producing these compounds. The structure of 10,11-dehydro-epothilone D is as follows.
[783]
[784] [454] In another embodiment, the present invention provides a method for making any 10,11-dehydro-epothilone analog by inactivating the ER domain of extender module 5 of epothilone PKS that produces corresponding corresponding epothilones. to provide.
[785] In another embodiment, a strain is produced that produces 10,11-dehydroepothilone D by inactivating the enoyl reductase (ER) domain of extender module 5. In one embodiment, ER inactivation is achieved by altering two glycines (-Gly-Gly-) in the NADPH binding zone to alanine and serine (-Ala-Ser-). For position-directed mutations, a 2.5 kb BbvCi-HindIII fragment (subclone of the epoD gene from cosmid pKOS35-70.4) from plasmid pKOS39-118B was cloned into pLitmus28 as pTL7 used as template. Oligonucleotides for introducing a mutation from -Gly-Gly- to -Ala-Ser- into the NADPH binding domain are as follows:
[786] TLII-22, 5'-TGATCCATGCTGCGGCCG CTA GCGTGGGCATGGCCGC (SEQ ID NO: 61).
[787] TLII-23, 5'-GCGGCCATGCCCACGC TAG CGGCCGCAGCATGGATCA (SEQ ID NO: 62).
[788] PCR clones containing substituents were identified by sequencing, digested with restriction enzyme Dra I and treated with shrimp alkaline phosphatase. Large fragments of each clone were then conjugated to kanamycin resistance and galK gene (KG or kan-gal ) cassettes to create delivery plasmids. By using this transfer plasmid it was transformed by the epothilone D mikso producer Rhodococcus glass tooth K111-72.4.4 or K111-40-1 electrophoresis. Transformants were screened to screen kanamycin-sensitive, galactose-resistant survivors to identify clones from which the KG gene was removed. KG clearance was confirmed and the desired gene replacement by recombinant strain was performed by PCR. Recombinant strains were prepared in CTS medium (Cazette, 5 g / L; MgSo ₄ , 2 g / L; L-alanine, 1 mg / L; L-serine, 1 mg / L; glycine, 1 mg / L; and HEPES buffer , 50 mM) After fermentation in a flask with 50 mL and 2% XAD-16 for 7 days, 10,11-dehydro-epothilone D was eluted from XAD resin with 10 mL methanol.
[789] I. Production of Oxazole-containing Epothilones by Fermentation
[790] [457] In one embodiment, the present invention relates to oxazole-containing epothilones by fermenting an epothilone producing strain provided by the present invention, such as Sorangium cellulose strain or myxococcus strain, in a medium supplemented with L-serine. And the thiazole portion of the corresponding epothilone is replaced by oxazole.
[791] [458] To illustrate this aspect of the invention, the batch medium (2.3mM), except that one to L- serine is present in the amount of the 11x, 51x, 101x and 201x based serine concentration mikso glass tooth strain Lactococcus K111-40.1 or K111-72.4.4 cultures were fermented according to the method of Example 3. Batch medium-containing 50 mL cultures were thus: 20 g / L XAD-16; 5 g / L kaziton; 2g / L MgSO ₄ ; 7 mL / L methyl oleate; And a 4 mL / L mirya element solution, and a filter-sterilized 1.25 M solution of L-serine was added at an appropriate concentration. The batch titers observed in basal medium were Epo C: 0.4 mg / L, Epo D: 2 mg / L, Epo H1 (C analog of oxazole). Undetectable, and Epo H2 (D analogue of oxazole); 0.02 mg / L. Increasing serine concentrations reduced epoC and epoD concentrations (to almost undetectable levels of 51 × supplementation). Thus, the batch titers in 51 × supplementation of L-serine in basal medium were: Epo C: 0.03 mg / L, Epo D: 0.05 mg / L, Epo H1: 0.2 mg / L, and Epo H2: 0 13 mg / L. The fed-batch protocol could increase the observed titer by about 10 times.
[792] J. Preparation of Epothilone Analogs
[793] [459] In one embodiment, the invention is produced by the recombinant epothilone PKS enzyme of the invention, wherein (i) the specificity of extender module 1 NRPS is changed from cysteine to another amino acid; (ii) the loading domain is changed to NRPS or CoA ligase; Or (iii) both (i) and (ii) epothilones and epothilone derivatives. This example illustrates how this recombinant epothilone PKS enzyme of the invention is prepared; References cited in this example are listed at the end of this example and are referenced by citation numbers.
[794] [460] Epothilones contain cyclized amino acid cysteine and are oxidized to form thiazoles. Two other amino acids, serine and threonine, can undergo similar cyclization and oxidation to produce oxazole and methyloxazole, respectively. For example, the oxazoles and methyloxazoles of epothilone D are as follows:
[795]
[796] In order to prepare an epothilone analogue by oxazole or methyloxazole, extender module 1 and NRPS module can be manipulated. The NRPS module, which extends the growing molecule, includes domains that activate amino acids, adenylation domains, PCP or peptidyl carrier protein domains that bind amino acids to NRPS, and condensation domains that condense amino acids to carboxyl groups of growing molecules to form peptide bonds. It consists of at least (5,7). A recognition sequence for determining the specificity of an amino acid is found between the adenylation domain, in particular the A4 and A5 consensus sequences (4). Analysis of this band showed that key amino acids in this protein band can predict which amino acids will be used by NRPS (2,8). Experiments were conducted in which the complete NRPS adenylation zone was exchanged for another, resulting in hybrid NRPS with amino acid specificity of the new adenylation domain (6,9). Experiments using smaller bands of adenylation bands, such as between the A4 and A5 consensus sequences, have been reported. In one embodiment, from the adenylate biotinylated domain from epoB A4 and A5 cone bleomycin gene cluster, using a band, and serine from the vibF and blmVII the band between Census band, using a threonine blmVI NRPS-4 The hybrid PKS of the present invention is produced by substituting the band (3).
[797] [462] Recent experiments suggest that condensation domains may detect inaccurate amino acid attachments to PCPs (1). Once an incorrect amino acid is detected, the condensation reaction is less efficient. To avoid this, in addition to altering the adenylation domain, one can alter the specificity of the adenylation domain and bring in a cognate condensation domain to manipulate fully active NRPS.
[798] [463] The present invention also provides a recombinant epothilone PKS enzyme that produces 16 desmethyl derivatives in the form of oxazole and methyloxazole in the epothilones. Such enzymes are made by altering the AT domain of extender module 2, epoC, from methylmalonyl specific to malonyl specific. AT domains usable for making this construct include extender modules 5 and 9 of the epothilone cluster and extender modules 2 and 4 from the soraphen gene cluster.
[799] [464] The present invention also provides a recombinant PKS enzyme in which the EpoA protein is substituted by NRPS. The present invention also provides novel epothilones produced by such enzymes. Epothilone biosynthesis is initiated by loading malonate on the loading module, ACP of EpoA. This malonate is decarboxylated by the KS domain and then transferred to the EpoB, NRPS module as an acetyl moiety. If the module is acted by EpoB, the compound obtained is 2-methylthiazole.
[800] [465] To make analogs with amino acids attached at the 2 position of thiazole , deletion of epoA and insertion of NRPS module is required. Any NRPS module is also available; However, to achieve the most conservative modifications, one can use an NRPS module that replaces epoA , which naturally communicates with the downstream NRPS module. In addition to the Phu, NRPS substituted epoA is a loading module and therefore does not require a condensation domain. This can be accomplished by taking the extender NRPS module, removing the condensation domain, or using NRPS that lacks a condensation domain because it is naturally a loading module. Exemplary loading NRPS module is derived from a Lactococcus mikso glass tooth, it will from safB using alanine.
[801] [466] As in the production mikso Lactococcus glass tooth strains containing loading modules in safB epoA position, it is possible to determine the optimum boundaries of the new module and epoB. Linker zones between PKS proteins are often very important for "communication" between these proteins. Three different strains can be constructed to test the optimal linker. First, the fusion of the ACP domain of the loading domain EpoA the adenylate biotinylated domain of safB. This construct requires that Apo of EpoA act as a PCP. Although the PCP and ACP domains are functionally similar, they do not exhibit a high degree of sequence homology and may be limited by their recognition and binding. The second construct fuses the last few amino acids downstream of EpoA downstream of the PCP domain of the SafB loading module, thereby providing EpoB with the necessary linker band of the hybrid roddy module required for "communication". Finally, a fusion of the SafB loading module with EpoB is prepared. Since SafB consists of two modules, taking all of the loading modules of SafB and fusion them directly to EpoB provides a fusion protein that is best suited for communication between the SafB loading module and EpoB.
[802] [467] Once a SafB or other loading NRPS domain is used to replace 2-methyl on the thiazole of epothilone with any amino acid, any new amino acid can be used to initiate the synthesis of the epothilone analog. Changes can occur. A list of available amino acids and their corresponding NRPS modules available for such exchanges is provided in Challis et al. (2).
[803] [468] epothilone containing mikso Lactococcus glass tooth strain K111-32.25, or epothilone gene containing the gene, and epoK gene is functional product that does not produce the mikso Lactococcus glass tooth strain K111-40-1, or All such substitutions can occur in any other epothilone producing strain or Soranium cellulose island of the invention. In Myxococcus, appropriate constructs can be made in plasmids using galK and kanamycin selectivity, which are used to replace wild-type genes with genetically engineered ones. For example, the substitution of NRPS with NRPS-specific serine in K111-40-1 produces 2-methyl-oxazole derivatives of epothilone D and 2-methyl-oxazole derivatives of epothilone C as main and by-products, respectively. It is expected to be. The structure of these compounds is as follows:
[804]
[805] Substitution of NRPS with NRPS specific threonine in K111-40-1 is expected to produce the following compounds as main and by-products, respectively:
[806]
[807] Substitution of K111-40-1 NRPS with glycine, alanine, glutanic acid, aspartic acid, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, arginine, valine, and tyrosine-specific NRPS It is expected that the following compounds will be made as by-products:
[808]
[809] Wherein R 'corresponds to a specific side chain among amino acids (e.g., for glycine in the general amino acid formula NH ₂ -CHR'COOH, R' is H, methyl for alanine, and so on).
[810] [471] Substitution of K111-40-1 NRPS with proline-specific NRPS is expected to produce the following main and by-products:
[811]
[812] [472] The documents cited in this subsection are as follows.
[813] [473] 1. Belshaw et al. 1999. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis Science. 284: 486-9.
[814] [474] 2. Challis et al. 2000. Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains Chem Biol. 7: 211-24.
[815] [475] 3. Du et al. 2000. The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase Chem Biol. 7: 623-42.
[816] [476] 4. Konz et al. 1999. How do peptide synthetases generate structural diversity Chem Biol. 6: R39-48.
[817] [477] 5. Marahiel et al. 1997. Modular peptide synthetases involved in nonribosomal peptide synthesis Chem. Rev. 97: 2651-2673.
[818] [478] 6. Schneider et al. 1998. Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping Mol Gen Genet. 257: 308-18.
[819] [479] 7. Stachelhaus et al. 1995. Modular structure of peptide synthetases revealed by dissection of the multifunctional enzyme GrsA J Biol Chem. 270: 61639.
[820] [480] 8. Stachelhaus et al. 1999. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases Chem Biol. 6: 493-505.
[821] [481] 9. Stachelhaus et al. 1995. Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains Science. 269: 69-72.
[822] Example 12
[823] Biological activity
[824] 10,11-dehydroepothilone D was screened for anticancer activity in four different human tumor cell lines using a sulforhodamine B (SRB) assay. 10,11-dehydroepothilone D showed inhibitory activity against all four cell lines with an inhibitory effect of IC ₅₀ s in the range of 28 nM to 40 nM. The mechanism of action was measured by cell-based tubulin polymerization, which revealed that this compound promotes tubulin polymerization. Human cancer cell lines MCF-7 (breast), NCI / ADR-Res (breast, MDR), SF-268 (glioma), NCI-H460 (lung) were obtained from the National Cancer Institute. These cells were maintained in 37% 5% CO ₂ -humid atmosphere in RPMI 1640 medium (Life Technology) supplemented with 10% fetal bovine serum (Hyclone) and 2 mM L-glutamine.
[825] [483] Cytotoxicity of 10,11-dehydroepothilone D was measured by SRB analysis (Skehan et al . , J. Natl. Cancer Inst . 82: 1107-1112 (1990)-incorporated herein by reference). Cultured cells were trypsinized, counted and diluted to the following concentrations per 100 μL with medium: MCF-7, 5000; NCI / ADR-Res, 7500; NCI-H460, 5000; And SF-268, 7500. Cells were seeded at 100 μL / well in 96-well microtiter plates. After 20 hours, 100 μL of 10,11-dehydroepothilone D (1000 nM to 0.001 nM in culture medium medium) was added to each well. After incubation with the compound for 3 days, the cells were fixed with 100 μL of 10% trichloric acid (“TCA”) for 4 to 1 hour and then stained with 0.2% SRB / 1% acetic acid for 20 minutes at room temperature. Unbound dye was rinsed with 1% acetic acid and then bound SRB was extracted using 200 μL of 10 mM Tris base. The amount of dye bound was measured at OD 515 nm, which is related to the total cellular protein content. The data was then analyzed using the Kaleida Graph program and the IC ₅₀ calculated. Chemically synthesized epothilones D were tested as compared to each other.
[826] [484] For tubulin polymerization assays, MCF-7 cells were incubated in a 35 mm-culture dish until compacted and then 0 with 1 μM of 10,11-dehydroepothilone D or epothilone D at 37 ° C. Treatment was for 1 or 2 hours. (See Giannakakou et al. , J. Biol. Chem. 271 : 17118-17125 (1997); Int. J. Cancer 75: 57-63 (1998).). After washing the cells with 2 mL of PBS without calcium or magnesium, cells were washed with 300 μL of lysis buffer (20 mM Tris, pH 6.8, 1 mM MgCl _2, 2 mM EGTA, 1% Triton X-100, plus protease inhibitor) And dissolved for 5-10 minutes. Cells were scraped and lysates were transferred into 1.5 ml Eppendorf tubes. The lysates were centrifuged at 18000 g for 12 minutes at room temperature. Soluble or unpolymerized dskg was separated from the supernatant containing (cytosolic) tubulin from pellets containing insoluble or polymerized tubulin and transferred to a new test tube. The pellets were then resuspended in 300 μL of lysis buffer. Tubulin polymerization changes in cells were measured by analyzing the aliquots of the eastern blood of each sample by SDS-PAGE followed by immunoblotting with anti-tubulin antibody (Sigma).
[827] [485] Some experiments show that 10,11-dehydroepothilone D (named "Epo490") has an IC ₅₀ in the range of 28 nM to 40 nM for four different human tumor cell lines.
[828] Cell lineEpoD (nM)Epo490 (nM)N = 3N = 2 MCF-21 ± 1028 ± 8 NCI / ADR40 ± 1235 ± 9 SF-26834 ± 840 ± 5 NCI-H46030 ± 234 ± 1
[829] Bovine polymerization analysis revealed that 10,11-dehydroepothilone D had the same mechanism of action as epothilone D. In MCF-7 cells, 10,11-dehydroepothilone D strongly promoted tubulin polymerization with kinetic and effects similar to epothilones under the conditions tested. Other compounds of the invention can also be tested in a similar manner by substituting the compounds of the invention with 10,11-dehydroepothilone D.
[830] Example 13
[831] Oxazole derivatives
[832] This example illustrates the modification of the type of epothilone compound produced by the host cell using fermentation conditions. By enriching the host cell with an excess of serine, compounds commonly produced by the host cell are modified in a manner that is good for producing oxazole counterparts. For example, cells that primarily produce a compound of Formula V or compounds can be made to produce an oxazole corresponding compound corresponding to the compound of Formula VI.
[833] [487] In one embodiment, the production of epothilone C and a strain of Rhodococcus mikso glass tooth strain K111-40-1 which mainly produce D, epothilone C and D of the corresponding oxazole compound, epothilone H1 and H2 Can be made to increase significantly. Strain K111-40-1 (PTA-2712) was deposited November 21, 2000 in the American Type Culture Collection (“ATCC”), 10801 University Blvd., Manassas, VA, 20110-2209 USA. Strains K111-40-1 were cultured in media with or without supplemental serine. The final concentrations of the components in the unreinforced medium were as follows: hydrolyzed casein (pancreatic digest, marketed under the brand name Casitone by Difco 0, 5 g / L; MgSO ₄ · 7H ₂ O, 2 g / L; XAD -16, 20 g / L; trace element solution 4 mL / L; methyl oleate 7 ml / L; and Hepes buffer, 40 mM (titrated to pH 7.6 with KOH) The trace element solution comprises: concentrated sulfuric acid H ₂ SO ₄ , 10 mL / L; FeCl ₃ 6H ₂ O, 14.6 g / L; ZnCl ₂ 2.0 g / L; MnCl ₂ 4H ₂ O, 1.0 g / L; CuCl ₂ 2H ₂ O, 0.42 g / L H ₂ BO ₃ , 0.31 g / L; CaCl ₂ · 6H ₂ O 0.24 g / L; and Na ₂ MoO ₄ · 2H ₂ O 0.24 g / L.The basal concentration of serine was taken at 4.82% w / w, This is the value measured by Difco 'amino acid analysis of a specific lot of Casitone As a result, the basal serine concentration was 2.3 mM and this value was calculated from the final concentration of Casitone at an average of 5 g / L. Medium contained 50 times higher serine, ie 117 mM.
[834] Cells were incubated at 30 ° C. for 120 hours on a 250 rpm coffin shaker. The compounds produced by the cell strain during the fermentation were extracted by capturing the resin, the resin was washed once in water, and the compound in the resin was extracted for 30 minutes in 20 mL of methanol. Samples were analyzed using HPLC and mass spectroscopy.
[835] [489] Assays of the compounds produced by the cells resulted in a 50-fold increase in serine concentration, resulting in a 30-fold increase in epothilone H1 production (0.12 mg / l) over the amount produced by cells cultured in serine-enriched media. L), the production of epothilone H2 was found to be increased fivefold. Note that the cells produced almost unsearchable amounts of epothilones C and D (<50 μg / L).
[836] [490] A method for producing an oxazole compound from a host cell that would normally produce a thiazole-containing counterpart would be provided by increasing the oxazole-containing compound and decreasing the thiazole-compound simultaneously due to serine supply. It became. For example, the recombinant for making 9-oxo epothilone D (as described in subpart C of Example 11) is 17-des (2-methyl-4-, an oxazole-compatible compound of 9-oxo-epothilone D It can be cultured under similar conditions to make thiazole) -17- (2-methyl-4-oxazolyl) -9-oxo-epothilone D. Likewise, other recombinant constructs of the present invention, including those described by Example 11, can also be incubated with excess serine feed to produce the corresponding oxazole compounds.
[837] Example 14
[838] Microbial transformation from C-21 methyl to C-21 hydroxymethyl
[839] This example illustrates the microbial transformation of C-21hydroxymethyl from C-21methyl of a compound of formula (I) in which Ar is:
[840]
[841] PCT Disclosure No. Amino coke other Outlet trophy, as described in WO 00/39276 car (Amycollata autotrophica) ATCC 35203 or evil Martino My process sp. Frozen vials of strain PTA-XXX (about 2 ml) were used to inoculate 1500 mL flasks containing 100 mL of medium. The nutrient medium consists of 10 g of dextrose, 10 g of malt extract, 10 g of yeast extract, and 1 g of peptone per liter of deionized water. The nutrient cultures were incubated for 3 days at 28 ° C. on a rotary shaker running at 250 rpm. 1 mL of the resulting culture was added to each of the 62 500 mL flasks containing the transformation medium having the same composition as the nutrient medium. Cultures were incubated at 28 ° C., 250 rpm for 24 hours. Appropriate compounds of the present invention were dissolved in 155 mL of ethanol, 155 mL of ethanol and the solution was divided into 62 flasks. The flask was then placed back on the shaker and incubated further at 28 ° C., 250 rpm for 43 hours. The reaction culture was then processed to recover the 21-hydroxy counterpart of the starting compound.
[842] Example 15
[843] Epoxidization with EpoK
[844] This example illustrates the enzymatic epoxidation of a compound of formula (I), a desoxy compound of the invention, wherein R ⁸ and R ¹⁰ together form a carbon carbon double bond. As described in PCT Publication WO00 / 31247, referenced herein, polyhistidine tags (his tags) were used to express epoK gene products as fusion proteins in E. coli and then to purify them. The reaction was carried out in 100 μL volume, 50 mM Tris (pH7.5), 21 M spinach ferredoxin, spinach ferredoxin 0.132 units: NADP ⁺ oxidoreductase, glucose-6-phosphate dehydrogenase 0.8 unit, 1.4 mM NADP, And 7.1 mM glucose-6-phosphate, 100 μM or 200 μM desoxy compounds of the invention, and 1.7 μM amino terminal histidine labeled EpoK or 1.6 μM carboxy terminal histidine labeled EpoK. The reaction was stopped by incubating at 30 ° C. for 67 minutes and then heating at 90 ° C. for 2 minutes. Insoluble material was removed by centrifugation and 50 μL of the supernatant containing the desired product was analyzed by LC / MS.
[845] Example 16
[846] Chemical epoxidation
[847] This example illustrates the chemical epoxidation of a compound of formula (I) (desoxy compound of the invention) in which R ⁸ and R ¹⁰ together form a carbon carbon double bond. Dimethyldioxirane solution (0.1 M in acetone, 17 mL) was added dropwise to the deoxy compound solution of the invention in 10 mL of CH ₂ Cl ₂ at -78. The mixture was warmed to −50 ° C., maintained for 1 hour, then further dimethyldioxirane solution (5 mL) was added and the reaction continued again at −50 ° C. for 1.5 hours. The reaction was then dried at -50 ° C under N ₂ air stream. The product was purified by chromatography on SiO ₂ .
[848] Example 17
[849] ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E) -15-amino-3,7-dihydroxy-5,9-dioxo-12,13-epoxy-4,4,6,8,12,16-hexamethyl-17- (2-methylthiazole -4-yl) -16-heptadecanoic acid
[850]
[851] [494] First step. 9-oxoepothilone B. To a solution of 9-oxoepothilone D (505 mg) in 10 mL CH2Cl2 at -78 ° C was added dropwise a solution of dimethyldioxirane (acetone, 0.1M in 17 mL). The mixture was warmed to −50 ° C., held for 1 hour, then further dimethyldioxirane solution (5 mL) was added and the reaction continued at −50 ° C. for 1.5 hours. The reaction was then dried at -50 ° C under N ₂ air stream. The product was purified by chromatography on SiO ₂ .
[852] [495] Second step. ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -15-azido-3,7-dihydroxy-5,9-dioxo-12,13-epoxy-4,4,6, 8,12,16-hexamethyl-17- (2-methylthiazol-4-yl) -16-heptadecanoic acid.
[853] A solution of 9-oxoepothilone B (2.62 g) and sodium azide (0.49 g) in 55 mL of degassed tetrahydrofuran / water (10: 1 v / v) was subjected to tetrakis (triphenylforce) in an argon atmosphere. Pin) palladium (0.58 g). The mixture was kept at 45 ° C. for 1 hour, then diluted with 50 mL of water and extracted with ethyl acetate. The extract was washed with brine, dried over Na 2 SO 4, filtered and evaporated. This product was purified by chromatography on SiO ₂ .
[854] [496] Third step. ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -15-amino-3,7-dihydroxy-5,9-dioxo-12,13-epoxy-4,4,6,8 , 12,16-hexamethyl-17- (2-methylthiazol-4-yl) -16-heptadecanoic acid. ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -15-azido-3,7-dihydroxy-5,9-dioxo in 15 mL THF / water (10: 1 v / v) A -12,13-epoxy-4,4,6,8,12,16-hexamethyl-17- (2-methylthiazol-4-yl) -16-heptadecanoic acid (565 mg) solution under argon, Treatment with trimethylphosphine 1.0M solution in toluene (3 mL) at ambient temperature. This mixture was concentrated and then the product was purified by flash chromatography on SiO ₂ .
[855] Example 18
[856] ( 4S, 7R, 8S, 9R, 13R, 14S, 16S) -13,14-epoxy-4,8-dihydroxy-2,6,10-trioxo-5,5,7,9,13-pentamethyl-16- (1- (2-methylthiazole- 4-yl) propen-2-yl) -1-aza-11-cyclohexadecene
[857]
[858] [497] ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -15-amino-3,7-dihydroxy in acetonitrile / dimethylformamide (20: 1 v / v, 150 mL) -5,9-dioxo-12,13-epoxy-4,4,6,8, 12,16-hexamethyl-17- (2-methylthiazol-4-yl) -16-heptadecanoic acid (540 mg ) Solution was cooled to 0 ° C. and treated sequentially with 1-hydrocybenzotriazole (0.135 g) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.5 g). The mixture was warmed to ambient temperature, held for 12 hours, diluted with water and extracted with ethyl acetate. The extract was washed sequentially with water, saturated NaHCO ₃ and brine, then dried over Na ₂ SO ₄ , filtered and evaporated. This product was purified by flash chromatography on SiO ₂ .
[859] Example 19
[860] ( 4S, 7R, 8S, 9R, 13Z, 16S) -4,8-dihydroxy-2,6,10-trioxo-5,5,7,9,13-pentamethyl-16- (1- (2-methylthiazol-4-yl) propene -2-yl) -1-aza-11-cyclohexadecene
[861]
[862] A solution of tungsten hexachloride (0.76 g) in tetrahydrofuran (20 mL) at 78 ° C. was treated with a 1.6 M solution of n-butyllithium in hexane (2.5 mL). This mixture was heated to ambient temperature for 20 minutes. A 13.8 mL portion of the obtained green solution was added to ( 4S, 7R, 8S, 9R, 13R, 14S, 16S ) -4,8-dihydroxy-13,14-epoxy-2,6,10 in 2 mL tetrahydrofuran at ambient temperature. -Trioxo-5,5,7,9,13-pentamethyl-16- (1- (2-methylthiazol-4-yl) propen-2-yl) -1-aza-11-cyclohexadecene (360 mg) was added to the solution. After 30 minutes, diluted with water and extracted with CH ₂ Cl ₂ . The extract was dried over Na ₂ S0 ₄ , filtered and evaporated. The product was purified by flash chromatography on SiO ₂ .
[863] Example 20
[864] ( 4S, 7R, 8S, 9R, 13R, 14S, 16S) -13,14-epoxy-4,8-dihydroxy-2,6,10-trioxo-1,5,5,7,9,13-hexamethyl-16- (1- (2-methylthia Zol-4-yl) propen-2-yl) -1-aza-11-cyclohexadecene
[865]
[866] [499] First step. ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -3,7-dihydroxy-5,9-dioxy-12,13-epoxy-4,4,6,8,12,16 Hexamethyl-15- (methylamino) -17- (2-methylthiazol-4-yl) -16-heptadecanoic acid. ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -15-amino-3,7-dihydroxy-5,9-dioxy-12,13-epoxy-4,4 in 10 mL methanol , 6,8,12,16-hexamethyl-17- (2-methylthiazol-4-yl) -16-heptadecenoic acid (540 mg) with 37% aqueous formaldehyde (1 mL), acetic acid (25 L) And sodium cyanoborohydride (100 mg). After 1 h, the mixture was treated with 1N HCl and diluted with ethyl acetate and water. The aqueous phase was extracted with ethyl acetate and the organic phases combined, then dried over Na ₂ SO ₄ , filtered and evaporated. The product was purified by flash chromatography on SiO ₂ .
[867] [500] The second step. ( 4S, 7R, 8S, 9R, 13R, 14S, 16S ) -13,14-epoxy-4,8-dihydroxy-2,6,10-trioxo-1,5,5,7,9,13 Hexamethyl-16- (1- (2-methylthiazol-4-yl) propen-2-yl) -1-aza-11-cyclohexadecene. ( 3S, 6R, 7S, 8R, 12R, 13S, 15S, 16E ) -3,7-dihydroxy-5,9-dioxo in acetonitrile / dimethylformamide (20: 1 v / v, 150 mL) -12,13-epoxy-4,4,6,8, 12,16-hexamethyl-15- (methylamino) -17- (2-methylthiazol-4-yl) -16-heptadecenoic acid (554 mg) solution was cooled to 0 ° C. and treated sequentially with 1-hydroxybenzotriazole (0.135 g) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.5 g). The mixture was warmed to ambient temperature for 12 hours, then diluted with water and extracted with ethyl acetate. The extract was washed sequentially with water, saturated NaHCO ₃ , and brine, then dried over Na ₂ SO ₄ , filtered and evaporated. The product was purified by flash chromatography on SiO ₂ .
[868] Example 21
[869] ( 4S, 7R, 8S, 9R, 13Z, 16S) -4,8-dihydroxy-2,6,10-trioxo-1,5,5,7,9,13-hexamethyl-16- (1- (2-methylthiazol-4-yl) Propen-2-yl) -1-aza-11-cyclohexadecene
[870]
[871] A solution of tungsten hexachloride (0.76 g) in tetrahydrofuran (20 mL) was treated with 1.6 M n-butyllithium in hexane (2.5 mL) at 78 ° C. This mixture was warmed to room temperature for 20 minutes. A 13.8 mL portion of the obtained green solution was added to ( 4S, 7R, 8S, 9R, 13R, 14S, 16S ) -13,14-epoxy-4,8-dihydroxy-2,6, in 2 mL of tetrahydrofuran at ambient temperature. 10-trioxo-1,5,5,7,9,13-hexamethyl-16- (1- (2-methylthiazol-4-yl) propen-2-yl) -1-aza-11- Add to cyclohexadecene (370 mg). After 30 minutes, the reaction was cooled to 0 ° C. and treated with saturated NaHCO ₃ (10 mL). The mixture was diluted with water and extracted with CH ₂ Cl ₂ . The extract was dried over Na ₂ S0 ₄ , then filtered and evaporated. This product was purified by flash chromatography on SiO ₂ .
[872] Example 22
[873] Liposome composition
[874] [502] This example illustrates a liposomal composition containing 9-oxo-epothilone. A mixture of lipid and 9-oxo-epothilone D was dissolved in ethanol and the solution was dried under reduced pressure to dry as a thin film. The resulting lipid film was hydrated by addition of an aqueous phase and the particle size of the epothilone-derivative containing liposomes was adjusted to the desired range. Preferably the average particle diameter is less than 10 microns, more preferably from about 0.5 to about 4 microns. Particle size can be determined by, for example, mills (such as air-jet mills, ball mills, or vibrator mills), microprecipitation, spray-drying, lyophilization, high pressure homogenization, recrystallization from supercrystalline solvents, or by preserving an aqueous suspension of liposomes. By injection through a series of membranes (such as polycarbonate membranes) having a uniform pore size of, it can be reduced to a desired level. In one embodiment, the liposome composition contains: a compound of the present invention (1.00 mg); Phosphatidylcholine (16.25 mg); Cholesterol (3.75 mg); Polyethylene glycol derived distearyl phosphatidylethanolamine (5.00 mg); Lactose (80.00 mg); Citric acid (4.20 mg); Tartaric acid (6.00 mg); NaOH (5.44 mg); Water (amount equivalent to 1 mL). In another embodiment, the liposome composition comprises: Compound of the Invention (1.00 mg); Phosphatidylcholine (19.80 mg); Cholesterol (3.75 mg); Distearyl phosphatidylcholine (1.45 mg); Lactose (80.00 mg); Citric acid (4.20 mg); Tartaric acid (6.00 mg); NaOH (5.44 mg); Water (amount equivalent to 1 mL). In another embodiment, the liposome composition contains: Compound of the Invention (1.00 mg); 1-palmitoyl-2-olein-sn-glycero-3-phosphocholine (17.50 mg); 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol, Na (7.50 mg); Lactose (80.mg); Citric acid (4.20 mg); Tartaric acid (6.00 mg); NaOH (5.44 mg); Water (amount equivalent to 1 mL). Compositions containing other compounds of the invention are prepared using conditions similar to those described above.
[875] Example 23
[876] Polyglutamic Acid Conjugate
[877] This example describes the preparation of a poly-glutamic acid-21-hydroxy-9-oxo-epothilone D conjugate. Poly (1-glutamic acid) (“PG”) sodium salt (MW 34K, Sigma, 0.35 g) was dissolved in water. The pH of the aqueous solution was adjusted to 2 with 0.2 M HCl. The precipitates were collected, dialyzed with distilled water, and lyophilized to obtain 0.29 g of PG. To this PG solution (75 mg, repeat unit FW 170, 0.44 mmol) in dry DMF (1.5 mL) add 20 mg of 21-hydroxy-9-oxo-epothilone D, 15 mg of dicyclohexylcarbodiimide ("DCC") And trace amount of dimethylaminopyridine were added. The reaction was allowed to proceed for 4 hours at room temperature or until complete reaction by thin layer chromatography. The reaction mixture was placed in chloroform and the resulting precipitate was collected and dried in vacuo to yield approximately 65 mg of PG-21-hydroxy-9-oxo-epothilone D conjugate. The weight ratio of the compound of the present invention to PG in the starting material was varied to obtain various concentrations of 21-hydroxy-10,11-dehydroepothilone D as polymer conjugate. Conjugates of other compounds of the invention are prepared using conditions similar to those described above.
[878] Example 24
[879] Intravenous composition
[880] This example describes an intravenous composition of 9-oxo-epothilone D. This composition contains 10 mg / mL of 9-oxo-epothilone D in a vehicle containing 30% propylene glycol, 20% Creomophor EL, and 50% ethanol. Vehicle was weighed with ethanol (591.8 g) into a beaker containing a stir bar; Creomophor El (315.0 g) was added to this solution and mixed for 10 minutes; Propylene glycol (466.2 g) is added to the solution and prepared by mixing again for 10 minutes. 9-oxo-epothilone D (1 g) was placed in a 1 L volumetric flask containing 400-600 mL of vehicle and mixed for 5 minutes. 10,11-dehydroepothilone was added to the solution, and the volume was adjusted to 1 L; Again mixed for 10 minutes and filtered through a 0.22μm Millipore Millipak filter. The resulting solution was filled with a volume of 5.15 mL / vial to a sterile 5 mL vial using a peristaltic pump with meter. Filled vials were immediately stowed and crimped.
[881] [505] This vial containing 10 mg / mL of 9-oxo-epothilone D was diluted in normal saline or 5% dextrose solution and administered in a non-PVC, non-DEHP bag for administration to a patient. made. The product was injected to deliver the desired dosage for a period of 1 to 6 hours.
[882] [506] In one embodiment, the composition of the present invention was diluted 20-fold with sterile saline prior to intravenous injection. Final injection concentrations were 0.5 mg / mL of the compound of the present invention, propylene glycol 1.5%, Chremophor EL 1%, and ethanol 2.5%, which were injected for 1 to 6 hours to deliver the desired dose.
[883] Intravenous compositions containing other compounds of the present invention may be prepared and used in a similar manner.
[884] Example 25
[885] Cremophor Toxicity Pretreatment
[886] [508] This example is Cremophor Describe how to pretreat toxicity. Cremophor Compositions of the compounds of the present invention containing can cause toxicity to patients. Pretreatment with steroids was used to prevent anaphylaxis. Any suitable corticosteroid or corticosteroid and a complex of H ₁ antagonists and / or H ₂ antagonists are available. In one embodiment, Cremophor One hour prior to treatment with a compound of the present invention in a composition containing N, 50 mg of diphenylhydramine and 300 mg of cimetidine are orally administered to the patient. In another embodiment, Cremophor At least one and a half hours prior to treatment with a compound of the present invention in a composition containing N, 20 mg of dexamethasone is administered to the patient in advance. In another embodiment, Cremophor At least one and a half hours prior to treatment with the compound of the present invention in a composition containing N, 50 mg of diphenylhydramine, 300 mg of cimetidine and 20 mg of dexamethasone are administered intravenously in advance. In another embodiment, Cremophor At least one and a half hours before treatment with a compound of the present invention in a composition containing diphenylhydramine (5 mg / kg, iv), taking into account the weight of the patient; Cimetidine (5 mg / kg, iv); And dexamethasone (1 mg / kg, im).
[887] [509] All scientific and patent literature cited in the text are referred to in the text. Although the present invention has been described by way of examples and detailed descriptions, those skilled in the art will be aware of the above detailed descriptions and examples without departing from the scope of the appended claims, which are presented for purposes of explanation and various other embodiments. Will understand.

权利要求:
Claims (47)
[1" claim-type="Currently amended] Two kinds of poly Kane Tide synthase (PKS: polyketide synthase) recombinant expression and containing a vector, to produce a poly Kane Tide synthesized by a PKS enzyme encoded on said vector, cis (Cystobacterineae) in tobak Te Linea suborder encoding the gene Recombinant host cell.
[2" claim-type="Currently amended] The host cell of claim 1, wherein said host cell is selected from the family Myxococcaceae .
[3" claim-type="Currently amended] The host cell of claim 1, wherein said host cell is selected from the family Cystobacteraceae .
[4" claim-type="Currently amended] The host cell of claim 2, wherein the host cell is selected from the genus selected from Angiococcus, Myxococcus and Corallococcus .
[5" claim-type="Currently amended] The host cell of claim 3, wherein the host cell is selected from the group selected from Cystobacter , Melttangium , Stigmatella , and Archangium .
[6" claim-type="Currently amended] The host cell of claim 4 selected from the genus Myxococcus .
[7" claim-type="Currently amended] 6. The host cell of claim 5 selected from the Stigmatella genus.
[8" claim-type="Currently amended] The host cell of claim 6 selected from Myxococcus stipitatus, Myxococcus fulvus, Myxocuccus xanthus , and Myxococcus virescens .
[9" claim-type="Currently amended] 8. The host cell of claim 7, wherein the host cell is selected from Stigmatella erecta and Stigmatella aurantiaca .
[10" claim-type="Currently amended] 9. The host cell of claim 8 which is Myxocuccus xanthus .
[11" claim-type="Currently amended] A method for producing polyketides in a host cell of Cystobacterineae subfamily, which does not naturally produce polyketides , wherein the host cell of claim 1 is expressed in a vector encoding the PKS gene encoded by the vector. Culturing under conditions in which the ketide is produced.
[12" claim-type="Currently amended] The recombinant host cell of claim 1, wherein the recombinant host cell produces an epothilone or an epothilone derivative.
[13" claim-type="Currently amended] The host cell of claim 10, wherein the host cell produces an epothilone or an epothilone derivative.
[14" claim-type="Currently amended] The host cell of claim 13, wherein the host cell produces Epothilones A, B, C and D.
[15" claim-type="Currently amended] The host cell of claim 14, which is Myxocuccus xanthus K111-32.25.
[16" claim-type="Currently amended] 15. The host cell of claim 14, wherein the host cell produces epothilones A and B as the main product and epothilones C and D as the byproducts.
[17" claim-type="Currently amended] 14. The host cell of claim 13, wherein epothilones C and D are produced as main products.
[18" claim-type="Currently amended] 18. The host cell of claim 17, which contains no epoK gene or does not fully express a functional epoK gene product.
[19" claim-type="Currently amended] 19. The host cell of claim 18, which is Myxocuccus xanthus K111-40.1.
[20" claim-type="Currently amended] 19. The host cell of claim 18, which is Myxocuccus xanthus K111-72.4.4.
[21" claim-type="Currently amended] The host cell of claim 13, wherein the coding sequence of the module of epothilone PKS contains the epothilone PKS gene altered by mutation, deletion or substitution.
[22" claim-type="Currently amended] 22. The host cell of claim 21, wherein said module is extender module 6.
[23" claim-type="Currently amended] 23. The host cell of claim 22, wherein said module lacks a functional ketoductase domain and produces 9-keto epothilone.
[24" claim-type="Currently amended] The host cell of claim 21, wherein said module is extender module 5.
[25" claim-type="Currently amended] The host cell of claim 24, wherein module 5 lacks a functional dehydratase domain and produces 13-hydroxy epothilone.
[26" claim-type="Currently amended] The host cell of claim 21, wherein said module is extender module 4.
[27" claim-type="Currently amended] 27. The host cell of claim 26, wherein said module lacks a functional ketoductase domain and produces 13-keto epothilone.
[28" claim-type="Currently amended] 22. The method of claim 21, wherein the module is extender module 2, the coding sequence of the ketocinase domain is changed by mutation to change the active site cysteine to another amino acid, and to produce an epothilone or epothilone derivative, Host cells in which equivalent compounds must be provided.
[29" claim-type="Currently amended] 29. The host cell of claim 28, which is Myxocuccus xanthus K190-132.1.1.2.
[30" claim-type="Currently amended] The host cell of claim 21, wherein said module is extender module 1.
[31" claim-type="Currently amended] 31. The host cell of claim 30, wherein said module is modified to bind amino acids other than cysteine.
[32" claim-type="Currently amended] The host cell of claim 21, wherein said module is a loading module.
[33" claim-type="Currently amended] The host cell of claim 32, wherein said module is substituted by a module that binds to an amino acid.
[34" claim-type="Currently amended] A diketide equivalent compound having the formula

Epothilone derivatives produced by incubation with:

Wherein R ⁷ is hydrogen or methyl and Ar is aryl selected from:

Wherein R is hydrogen, hydroxy, halogen, amino, C _1-5 alkyl, C _1-5 hydroxyalkyl, C _1-5 alkoxy and C _1-5 aminoalkyl.
[35" claim-type="Currently amended] The method of claim 1, wherein the enzyme is used to deliver a compound used for biosynthesis of polyketide to the cell, an enzyme for synthesizing a compound used for the synthesis of polyketide, and an enzyme for phosphopanthethenylating PKS. A host cell further comprising a heterologous gene that encodes an enzyme.
[36" claim-type="Currently amended] 36. The host cell of claim 35, wherein said enzyme is MatB.
[37" claim-type="Currently amended] 36. The host cell of claim 35, wherein said enzyme is MatC.
[38" claim-type="Currently amended] 36. The host cell of claim 35, wherein said enzyme is MtaA.
[39" claim-type="Currently amended] The method according to claim 13, wherein the epothilone or epothilone derivative is selected from the group consisting of a promoter from the Sorangium cellulosum epothilone PKS gene, a promoter from the myxothiazole biosynthesis gene, a promoter from the TA biosynthetic gene, a pilA promoter, A host cell characterized by being produced by PKS under the control of a promoter from a kanamycin resistance conferring gene, and a promoter selected from the So ce90 promoter.
[40" claim-type="Currently amended] A method of purifying epothilone from cells producing epothilone, the method culturing the cells in the presence of XAD resin, eluting epothilone from the resin, and performing solid phase extraction of the epothilone eluted from the resin. Then, chromatographically treating the epothilone obtained from said solid extract.
[41" claim-type="Currently amended] 37. The method of claim 36, further comprising a crystallization step.
[42" claim-type="Currently amended] Crystalline epothilone D.
[43" claim-type="Currently amended] A method for fermenting a Myxococcus host cell, comprising culturing the Myxococcus host cell in a liquid medium containing a fatty acid or oil as a carbon source.
[44" claim-type="Currently amended] 44. The method of claim 43, wherein the fermentation is fed-batch fermentation.
[45" claim-type="Currently amended] 44. The method of claim 43, wherein said Myxococcus host cell produces an epothilone or an epothilone derivative.
[46" claim-type="Currently amended] 46. The method of claim 45, wherein said host cell produces an epothilone derivative containing oxazole instead of thiazole, and said liquid medium comprises L-serine.
[47" claim-type="Currently amended] Isolated compounds having the formula:

Wherein R ¹ , R ² , R ³ , R ⁵ , R ¹¹ and R ¹² are each independently hydrogen, methyl or ethyl;
R ⁴ , R ⁶ and R ⁹ are each independently hydrogen, hydroxyl or oxo;
or
R ⁵ and R ⁶ together form a carbon carbon double bond;
R ⁷ is hydrogen, methyl or ethyl;
R ⁸ and R ¹⁰ both represent hydrogen or together represent a carbon carbon double bond or an epoxide;
Ar is aryl;
W is O or NR ¹³ where R ¹³ is hydrogen, C _1-10 aliphatic, aryl or alkylaryl.

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同族专利:

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公开号 | 公开日

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KR100832145B1|2008-05-27|

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AT309369T|2005-11-15|

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ES2254493T3|2006-06-16|

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IL151960D0|2003-04-10|

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NZ521788A|2007-04-27|

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WO2001083800A3|2003-04-10|

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CA2404938A1|2001-11-08|

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CA2404938C|2011-11-01|

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AU9519501A|2001-11-12|

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NZ546497A|2008-04-30|

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EP1652926A2|2006-05-03|

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PT1652926E|2009-12-03|

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AU2001295195B2|2007-02-01|

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WO2001083800A9|2003-09-12|

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CN100480389C|2009-04-22|

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CN1511192A|2004-07-07|

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KR20070092334A|2007-09-12|

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WO2001083800A8|2003-01-03|

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EP1320611A2|2003-06-25|

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DE60140087D1|2009-11-12|

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DE60114865T2|2006-07-27|

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ES2332727T3|2010-02-11|

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AT444368T|2009-10-15|

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EP1652926B1|2009-09-30|

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CY1109698T1|2014-08-13|

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MXPA02010565A|2004-05-17|

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EP1320611B1|2005-11-09|

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DK1652926T3|2010-02-15|

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EP1652926A3|2006-08-09|

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WO2001083800A2|2001-11-08|

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DE60114865D1|2005-12-15|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-04-28|Priority to US09/560,367

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2000-04-28|Priority to US09/560,367

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2000-09-14|Priority to US23269600P

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2000-09-14|Priority to US60/232,696

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2000-12-21|Priority to US25751700P

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2000-12-21|Priority to US60/257,517

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2001-02-13|Priority to US26902001P

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2001-04-03|Priority to US09/825,876

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2001-04-03|Priority to US09/825,856

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2001-04-03|Priority to US09/825,876

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2001-04-03|Priority to US09/825,856

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2001-04-13|Priority to US60/269,020

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2001-04-26|Application filed by 코산 바이오사이언시즈, 인코포레이티드

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2001-04-26|Priority to PCT/US2001/013793

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2003-04-26|Publication of KR20030032942A

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2008-05-27|Application granted

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2008-05-27|Publication of KR100832145B1

优先权:

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申请号 | 申请日 | 专利标题

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US09/560,367|US6410301B1|1998-11-20|2000-04-28|Myxococcus host cells for the production of epothilones|

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US09/560,367|2000-04-28|

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US23269600P| true| 2000-09-14|2000-09-14|

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US60/232,696|2000-09-14|

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US25751700P| true| 2000-12-21|2000-12-21|

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US60/257,517|2000-12-21|

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US26902001P| true| 2001-02-13|2001-02-13|

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US09/825,856|US6489314B1|2001-04-03|2001-04-03|Epothilone derivatives and methods for making and using the same|

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US09/825,876|2001-04-03|

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US09/825,856|2001-04-03|

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US09/825,876|US6589968B2|2001-02-13|2001-04-03|Epothilone compounds and methods for making and using the same|

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US60/269,020|2001-04-13|

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PCT/US2001/013793|WO2001083800A2|2000-04-28|2001-04-26|Heterologous production of polyketides|