Streptomyces avermitilis gene directing the ratio of b2:b1 avermectins

专利摘要:
The present invention includes a nucleotide sequence encoding the AveC gene product, which can be used to vary the ratio or amount of type 2: 1 avermectin produced in fermentation cultures of Streptomyces avermitilis . It relates to a nucleotide molecule. The invention further relates to mutant strains of Streptomyces avermitilis mutated to alter the ratio or amount of the type 2: 1 avermectin from which the vector, host cell, and aveC gene are inactivated or produced. will be.
公开号:KR20020029379A
申请号:KR1020027001807
申请日:2000-07-24
公开日:2002-04-18
发明作者:첸얀；구스타프슨클레이스；크레버앵크；민슐제레미스티븐；레일라드선아이；스투츠만-잉월킴조넬
申请人:실버스타인 아써 에이.；화이자 프로덕츠 인코포레이티드；
IPC主号:

专利说明:

STEPTOMYCES AVERMITILIS GENE DIRECTING THE RATIO OF B2: B1 AVERMECTINS}
[2] Avermectin
[3] Streptomyces species produce a wide variety of secondary metabolites, including avermectins, comprising eight series of related 16-membered macrocyclic lactones with potent antiparasitic and pesticidal activity. The eight distinct but closely related compounds are referred to as Ala, A1b, A2a, A2b, B1a, B1b, B2a and B2b. Compounds of the series "a" represent natural avermectins having a substituent at the C25 position (S) -tertiary-butyl, and compounds of the series "b" represent natural avermectins having a substituent at the C25 position isopropyl. Indicates. The designations "A" and "B" represent evermectin, wherein the substituents at the C5 position are methoxy and hydroxy, respectively. The number "1" represents avermectin with a double bond present at the C22 and C23 positions, and the number "2" represents avermectin with hydrogen at the C22 position and hydroxy at the C23 position. Of these related avermectins, the B1 type of avermectins are known to have the most effective pesticidal and pesticidal activity and are therefore the most preferred commercial avermectins.
[4] Avermectin and its production by aerobic fermentation of Streptomyces avermitilis strains are described in US Pat. Nos. 4,310,519 and 4,429,042. Biosynthesis of natural avermectin is believed to be initiated endogenously from CoA thioester analogs of isobutyric acid and S-(+)-2-methylbutyric acid.
[5] By combining strain improvement through random mutagenesis and the use of exogenously supplied fatty acids, avermectin analogs were effectively produced. Mutant strains of streptomyces avermitilis lacking branched chain 2-oxo acid dehydrogenase (bkd deficient mutants) can only produce avermectin when the fermentation is supplemented with fatty acids. Screening and isolation of mutant strains lacking branched chain dehydrogenase activity (eg Streptomyces Avermitilis, ATCC 53567) are described in EP 246103. Fermentation of the mutant strain in the presence of an exogenously supplied fatty acid produces only four avermectins corresponding to the applied fatty acid. Therefore, supplementing fermentation of Streptomyces avermitilis (ATCC 53567) with S-(+)-2-methylbutyric acid produces natural avermectins A1a, A2a, B1a and B2a; Supplementation of fermentation with isobutyric acid produces natural avermectin A1b, A2b, B1b and B2b; Supplementation of fermentation with cyclopentanecarboxylic acid produces four new cyclopentylavermectins A1, A2, B1 and B2.
[6] When supplemented with other fatty acids, new avermectins are produced. By screening over 800 potential precursors, more than 60 other novel avermectins have been identified (see, eg, Dutton et al., J. Antibiot ., 44 , 357-365, 1991) and [ Banks et al., Roy. Soc. Chem ., 147 , 16-26, 1994). In addition, the mutant strain of Streptomyces avermitilis lacking 5-O-methyltransferase activity produces essentially only the B analog avermectin. Thus, streptomyces avermitilis mutants lacking both branched chain 2-oxo acid dehydrogenase and 5-O-methyltransferase activity, only B avermectin corresponding to fatty acids applied to supplement fermentation. To produce. Therefore, supplementation of double mutants with S-(+)-2-methylbutyric acid produced only natural avermectin B1a and B2a; Supplementation with isobutyric acid or cyclopentanecarboxylic acid produces either natural avermectin B1b and B2b or novel cyclopentyl B1 and B2 avermectin, respectively. Supplementation of cyclohexane carboxylic acid to the double mutant strain is a preferred method for producing cyclohexyl avermectin B1 (doraramtin), a novel commercially important avermectin. The isolation and characterization of such double mutant strains (eg Streptomyces avermitilis (ATCC 53692)) is described in EP 276103.
[7] 2. Genes involved in avermectin biosynthesis
[8] In many cases, genes involved in the production of secondary metabolites, and genes encoding specific antibiotics, have been found in bundles on chromosomes. This is due, for example, to the Streptomyces polyketide synthase gene family (PKS) (see Hopwood and Sherman, Ann. Rev. Genet ., 24 , 37-66, 1990). Therefore, one method for cloning genes in the biosynthetic pathway has been to isolate drug resistant genes and test adjacent regions of the chromosome for other genes associated with the biosynthesis of specific antibiotics. Another method for cloning genes involved in the biosynthesis of important metabolites was supplementation of mutant strains. For example, a portion of the DNA library from an organism capable of producing a particular metabolite is introduced into a non-productive mutant, whereby the transformant producing the metabolite is screened. In addition, hybridization of aggregates using probes from other Streptomyces species has been used to identify and clone genes in biosynthetic pathways.
[9] Like genes required for biosynthesis of other Streptomyces secondary metabolites, genes involved in avermectin biosynthesis (the aveC gene) have been found in clusters on the chromosome. Numerous ave genes have been successfully cloned using vectors complementing the complementary Streptomyces avermitilis mutants blocked in avermectin biosynthesis. Cloning of such genes is described in US Pat. No. 5,252,474. In addition, Ikeda et al., J. Antibiot ., 48 , 532-534, 1995 also describe 4.82 Kb of Streptomyces avermitilis ( aveC ) as well as mutations in the aveC gene that produce single component B2a producers. Positioning of chromosomal regions comprising C22 and C23 dehydration steps for Bam HI fragments is described. Since an effective insect repellent compound ivermectin can be produced chemically from avermectin B2a, producers of a single component of the avermectin B2a are considered particularly useful for commercial production of ivermectin.
[10] For example, by identifying a mutant of the aveC gene that minimizes the complexity of avermectin production, such as a mutant that reduces the B2: B1 ratio of avermectin, the production and purification of commercially important avermectins will be simplified. will be.
[11] Summary of the Invention
[12] The present invention includes the complete aveC ORF (open-reading framework) of Streptomyces avermitilis , or a substantial portion thereof, and is located in the following complete position downstream from the aveC ORF original position of the Streptomyces avermitilis chromosome. It provides an isolated polynucleotide molecule lacking ORF. The isolated polynucleotide molecule of the invention is preferably the nucleotide sequence of the aveC ORF that is identical to the Streptomyces avermitilis AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or is present in Figure 1 (SEQ ID NO: 1) or a nucleotide sequence identical to a substantial portion thereof. The invention further provides an isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a degenerate variant thereof.
[13] The present invention is identical to the Streptomyces Abermitis AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or to the nucleotide sequence of the aveC ORF present in FIG. 1 (SEQ ID NO: 1) or a substantial portion thereof. Further provided are isolated polynucleotide molecules having a nucleotide sequence.
[14] The invention encodes a polypeptide having an amino acid sequence identical to the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) or a substantial portion thereof. There is further provided an isolated polynucleotide molecule comprising a nucleotide sequence.
[15] The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homologous gene product. In a preferred embodiment, the isolated polynucleotide molecule comprises a nucleotide sequence encoding the AveC homologous gene product from S. hygroscopicus , which homologous gene product comprises the amino acid sequence of SEQ ID NO: 4 or Including a substantial portion thereof. In a preferred embodiment, the isolated polynucleotide molecules of the invention encoding Streptomyces hygroscopius AveC homologous gene product comprise the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof.
[16] The invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence identical to the Streptomyces hygroscopicus nucleotide sequence of SEQ ID NO: 3. The invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the same polypeptide as the Streptomyces hygroscopius AveC homologous gene product having the amino acid sequence of SEQ ID NO: 4.
[17] The present invention provides a nucleotide sequence that is hybridized to a polynucleotide molecule having a nucleotide sequence of FIG. 1 (SEQ ID NO: 1) or SEQ ID NO: 3, or is complementary to the nucleotide sequence of FIG. 1 (SEQ ID NO: 1) or SEQ ID NO: 3 Further provided are oligonucleotides that hybridize to polynucleotide molecules having.
[18] The present invention further provides recombinant cloning vectors and expression vectors useful for cloning or expressing polynucleotides of the present invention comprising polynucleotide molecules comprising aveC ORF or aveC homologous ORF of Streptomyces avermitilis . In a non-limiting embodiment, the present invention provides plasmid pSE186 (ATCC 209604) comprising the entire ORF of the aveC gene of Streptomyces avermitilis. The present invention further provides transformed host cells comprising the polynucleotide molecules or recombinant vectors of the invention, and novel strains or cell lines derived therefrom.
[19] The invention further provides substantially purified or isolated recombinantly expressed AveC gene products or AveC homologous gene products, or substantial portions thereof as well as homologs thereof. The present invention provides for the production of a recombinant AveC gene product or AveC homologous product, under conditions conducive to one or more regulatory nucleotide sequences encoding the AveC gene product or AveC homologous gene product and controlling the expression of the polynucleotide molecule in a host cell. Culturing the transformed host cell with the recombinant expression vector comprising the polynucleotide molecule effectively associated with the element; And recovering the AveC gene product or AveC homologous gene product from this cell culture.
[20] The present invention is Streptomyces Abbe reumi subtilis aveC allele, plasmid pSE186 (ATCC 209604) of the AveC gene product-encoding sequences or a degeneracy variants, or Streptomyces Abbe reumi subtilis of the aveC as present in FIG. 1 Identical to the nucleotide sequence of the ORF (SEQ ID NO: 1) or a degenerate variant thereof, but further comprising one or more mutations thereby inactivating the wild type aveC allele to express a polynucleotide molecule comprising the mutated nucleotide sequence Nucleotide sequences that produce a different ratio or amount of avermectin than the cells produced by the cells of Streptomyces avermitilis strain ATCC 53692 expressing only the wild type aveC allele. Polynucleotides containing It further provides a molecule. In accordance with the present invention, the polynucleotide molecule can be used to produce novel identical strains exhibiting detectable changes in avermectin production compared to strains of Streptomyces avermitilis expressing only the wild type aveC allele. have. In a preferred embodiment, the polynucleotide molecule is useful for producing a novel identical strain that produces a reduced type 2: 1 ratio of avermectin compared to a Streptomyces avermitilis strain expressing only the wild type aveC allele. . In a further preferred embodiment, the polynucleotide molecule is useful for producing novel identical strains that produce increased levels of avermectin compared to Streptomyces avermitilis strains expressing only a single wild type aveC allele. In a further preferred embodiment, the nucleotide molecule is useful for producing novel strains of Streptomyces avermitilis in which the aveC gene is inactivated.
[21] The present invention provides a method for identifying mutations in aveC ORF of Streptomyces avermitilis that can alter the proportion and / or amount of avermectin produced. In a preferred embodiment, the present invention provides a method for treating a Streptomyces avermitilis strain wherein (a) the native aveC allele is inactivated so that a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product is introduced and expressed. Measuring the type 2: 1 ratio of avermectin produced by the cell; (b) the same Streptomyces avermitilis strain as in step (a) except expressing only the wild type aveC allele having the nucleotide sequence of the ORF (SEQ ID NO: 1) or the same nucleotide sequence as that of FIG. Measuring the type 2: 1 ratio of avermectin produced by the cells of the cell; And (c) a type 2: 1 ratio of avermectin produced by Streptomyces avermitilis cells in step (a) and Aber produced by Streptomyces avermitilis cells in step (b) By comparing the type 2: 1 ratio of mectin, the type 2: 1 ratio of avermectin produced by the Streptomyces avermitilis cells of step (a) is reduced to the Streptomyces avermitilis of step (b). Identifying a mutation in the aveC ORF that can alter the type 2: 1 ratio of avermectin if it differs from the type 2: 1 ratio of avermectin produced by the cells. Provides a method for identifying mutations in aveC ORF that can change the type 2: 1 ratio. In an aspect of the invention, the type 2: 1 ratio of avermectin is reduced by mutation.
[22] In a further preferred embodiment, the invention comprises a gene construct comprising (a) a nucleotide sequence that encodes a mutated AveC gene product in which the native aveC allele is inactivated, or which comprises a nucleotide sequence that encodes an AveC gene product Measuring the amount of avermectin produced by the cells of the Streptomyces avermitilis strain, wherein the polynucleotide molecule is introduced and expressed; (b) the same Streptomyces avermitilis strain as in step (a) except expressing only a single aveC allele having the nucleotide sequence of the ORF (SEQ ID NO: 1) or the same nucleotide sequence thereof Measuring the amount of avermectin produced by the cells of; And (c) comparing the amount of avermectin produced by the Streptomyces avermitilis cells of step (a) with the amount of avermectin produced by the Streptomyces avermitilis cells of step (b) Whereby the amount of avermectin produced by the Streptomyces avermitilis cells of step (a) is different from the amount of avermectin produced by the Streptomyces avermitilis cells of step (b), Mutations in aveC ORFs that can change the amount of avermectin produced or in gene constructs comprising such aveC ORFs, comprising identifying mutations in the aveC ORFs or gene constructs that can change the amount of avermectins It provides a way to check. In an embodiment of the invention, the amount of avermectin is increased by mutation.
[23] The present invention further provides recombinant vectors useful for preparing novel strains of Streptomyces avermitilis with altered avermectin production. For example, the present invention comprises mutated nucleotide sequences of the invention in the aveC gene region of the Streptomyces avermitilis chromosome to insert or replace the aveC allele with ORF or a portion thereof by homologous recombination. Provided are vectors that can be used to target any polynucleotide molecule. However, according to the present invention, a polynucleotide molecule comprising a mutated nucleotide sequence of the invention provided herein is also inserted into the Streptomyces avermitilis chromosome at a site other than the aveC gene, or the Streptomyces avermitili It may also act to modulate avermectin biosynthesis when maintained in episomes in human cells. Therefore, the present invention also encompasses polynucleotide molecules comprising the mutated nucleotide sequences of the present invention and may be used to insert or maintain polynucleotide molecules at sites of the Streptomyces avermitilis chromosome other than the aveC gene. Provides a vector that can be used. In a preferred embodiment, the invention inserts the mutated aveC allele into the Streptomyces avermitilis chromosome, thereby reducing the type 2: 1 ratio of avermectin compared to cells of the same strain expressing only the wild type aveC allele. Provided are gene replacement vectors that can be used to generate new strains of cells that produce cells.
[24] The present invention includes cells that express the mutated aveC allele and produce a proportion and / or amount of altered avermectin compared to cells of the same strain of Streptomyces avermitilis that express only the wild type aveC allele. It further comprises a method for preparing a novel strain of Streptomyces avermitilis. In a preferred embodiment, the invention expresses the mutated aveC allele and produces a type 2: 1 ratio of altered avermectin compared to cells of the same strain of Streptomyces avermitilis expressing only the wild type aveC allele. Type 2: 1 ratio of avermectin produced by cells of the Streptomyces avermitilis strain expressing the mutated allele compared to cells of the same strain expressing only the wild type aveC allele Transforming cells of a Streptomyces avermitilis strain having a vector with a mutated aveC allele encoding a gene product that changes the gene product; And selecting the transformed cells to produce a varied type 2: 1 ratio of avermectin compared to the type 2: 1 ratio produced by the cells of said strain expressing the wild type aveC allele. Provided are methods for preparing novel strains of Maises avermitilis. In a preferred embodiment, the type 2: 1 ratio of avermectin produced is reduced in the cells of the new strain.
[25] In a further preferred embodiment, the present invention provides an aber produced by cells of a Streptomyces avermitilis strain expressing a mutated aveC allele or gene construct compared to cells of the same strain expressing only the wild type aveC allele. Transforming the cells of the Streptomyces avermitilis strain with a vector having a genetic construct comprising a mutated aveC allele or aveC allele resulting in altered amounts of mectin ; And selecting the transformed cells producing a changed amount of avermectin compared to the amount of avermectin produced by the cells of the strain expressing only the wild-type aveC allele. Provided are methods for preparing novel strains of Streptomyces avermitilis comprising producing cells. In a preferred embodiment, the amount of avermectin produced is increased in the cells of the new strain.
[26] In a further preferred embodiment, the present invention provides a method of inactivating aveC alleles comprising transforming cells of a Streptomyces avermitilis strain expressing any aveC allele; And selecting transformed cells in which the aveC allele has been inactivated, thereby providing a method for preparing a novel strain of Streptomyces avermitilis comprising the inactivated aveC allele.
[27] The invention further provides novel strains of Streptomyces avermitilis comprising cells transformed with any polynucleotide molecule or vector comprising a mutated nucleotide sequence of the invention. In preferred embodiments, the wild-type in place of aveC allele, or in addition to addition and mutant contained the aveC cells expressing an allele, compared to the same strain which expresses only the wild-type aveC allele cells transformed type 2: 1 ratio of O Provided are novel strains of Streptomyces avermitilis that produce vermectin. In a more preferred embodiment, the cells of the novel strain produce a reduced type 2: 1 ratio of avermectin compared to cells of the same strain expressing only the wild type aveC allele. The novel strains are useful for large scale production of commercially desirable avermectins such as doramectin.
[28] Further preferred embodiments of the same strain of the invention is expressed in place of the specific aveC allele, or in addition to the addition mutated aveC allele, or a result, the wild-type aveC allele the aveC expression of the genetic composition comprising a allele only Provided is a novel strain of Streptomyces avermitilis comprising cells that produce a changed amount of avermectin compared to the amount of avermectin produced by cells of. In a preferred embodiment, the novel cells produce increased amounts of avermectin.
[29] In a further preferred embodiment, the invention provides a novel strain of Streptomyces avermitilis comprising cells in which the aveC gene has been inactivated. Such strains are useful for both the spectrum of different avermectins they produce compared to wild type strains, and the supplemental screening assays disclosed herein to determine whether target or random mutagenesis of the aveC gene affects avermectin production. Do.
[30] The invention Ah produced by which only the wild-type aveC allele expression does not express the mutated aveC allele Streptomyces Abbe reumi subtilis in the same strain cells expressing the aveC allele mutation as compared to the cells of the strain suberic Cells of a Streptomyces avermitilis strain expressing a mutated aveC allele encoding a gene product that changes the type 2: 1 ratio of mectin are cultured in medium under conditions that permit or induce the production of avermectin. step; And recovering the avermectin from the culture, further providing a method for producing avermectin. In a preferred embodiment, the type 2: 1 ratio of avermectin produced by the cells expressing the mutation is reduced. This method provides increased efficiency in producing commercially expensive avermectins such as doramectin.
[31] The present invention cells of the same strain expressing cost as compared with that only the wild-type aveC allele expressed without expression of the aveC allele or a genetic mutation configured Streptomyces Abbe reumi subtilis strain cells mutated aveC allele or genetic makeup Production of avermectin cells of a Streptomyces avermitilis strain expressing a mutated aveC allele or gene construct comprising the aveC allele resulting in producing an altered amount of avermectin produced by Culturing in medium under these acceptable or induced conditions; And recovering the avermectin from the culture, further providing a method for producing avermectin. In a preferred embodiment, the amount of avermectin produced by the cell expressing the mutation or gene construct is increased.
[32] The present invention is produced by a Streptomyces avermitilis strain expressing the mutated aveC allele of the present invention, and does not express the mutated aveC allele, but does not express the mutated aveC allele, but expresses only the wild type aveC allele Further provided are compositions of the novel avermectins produced in a reduced type 2: 1 ratio compared to the type 2: 1 ratio of avermectins produced by cells of the same strain of. The novel avermectin compositions may be present when produced in fermentation broth, or may be harvested therefrom and partially or substantially purified therefrom.
[1] FIELD OF THE INVENTION The present invention relates to compositions and methods for producing avermectin, and mainly relates to the field of animal hygiene. More particularly, the present invention comprises a nucleotide sequence encoding the AveC gene product, which can be used to regulate the ratio of type 2: 1 avermectin produced by fermentation culture of Streptomyces avermitilis . Polynucleotide molecules, and compositions and methods for screening these polynucleotide molecules. The present invention also relates to a novel mutant strain of Streptomyces avermitilis that regulates the ratio of type 2: 1 avermectin produced by mutating a vector, a transformed host cell, and the aveC gene.
[33] 1 depicts a DNA sequence comprising Streptomyces avermitilis aveC ORF (SEQ ID NO: 1), and an inferred amino acid sequence (SEQ ID NO: 2).
[34] Figure 2 depicts a plasmid vector pSE186 (ATCC 209604) comprising the entire ORF of the aveC gene of Streptomyces avermitilis.
[35] FIG. 3 shows the gene replacement vector pSE180 (ATCC 209605) comprising the ermE gene of Saccharopolyspora erythraea inserted into the aveC ORF of Streptomyces avermitilis .
[36] FIG. 4 shows Bam HI of avermectin polyketide synthase gene family derived from Streptomyces avermitilis with five overlapping cosmid clones identified (ie, pSE65, pSE66, pSE67, pSE68 and pSE69). Show the restriction map. Also shown is the relationship between pSE118 and pSE119.
[37] 5A-5D show HPLC analysis of fermentation products produced by Streptomyces avermitilis strains. Peak quantification was performed by comparing the standard amount of cyclohexyl B1. The residence time of cyclohexyl B2 was 7.4 to 7.7 minutes and the residence time of cyclohexyl B1 was 11.9 to 12.3 minutes. 5A depicts Streptomyces avermitilis strain SE180-11 with inactivated aveC ORF. 5B depicts Streptomyces avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604). 5C depicts Streptomyces avermitilis strain SE180-11 transformed with pSE187. 5D depicts Streptomyces avermitilis strain SE180-11 transformed with pSE188.
[38] FIG. 6 shows aveC ORF (SEQ ID NO: 2) of Streptomyces avermitilis , aveC homologous partial ORF of Streptomyces griseochromogenes (SEQ ID NO: 5) and Streptomyces hygros A comparison of inferred amino acid sequences encoded by aveC homologous ORF (SEQ ID NO: 4) from Copicus is shown. Valine residues in bold font are putative initiation sites for proteins. Conserved residues are shown in uppercase when homologous in all three sequences and in lowercase when homologous in two of the three sequences. The amino acid sequence comprises about 50% sequence identity.
[39] 7 is 564bp from a Streptomyces high-gloss nose kusu aveC homologous gene inserted into the Bsa AI / Kpn I site of the Abbe reumi subtilis aveC ORF Streptomyces; a Bsa AI / kpn I fragment of (base pair nucleotide pairs) The hybrid plasmid constructs that are included are shown.
[40] The present invention can be used to screen for mutated AveC gene products for the identification and properties of polynucleotide molecules having a nucleotide sequence encoding the AveC gene product of Streptomyces avermitilis, their effect on avermectin production. The construction of a novel strain of streptomyces avermitilis, and the discovery that certain mutated AveC gene products may reduce the ratio of B2: B1 avermectins produced by Streptomyces avermitilis. will be. As an example, the present invention relates to a polynucleotide molecule having the same nucleotide sequence as the Streptomyces Abermitis AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the ORF of FIG. 1 (SEQ ID NO: 1). And nucleotide molecules having mutated nucleotide sequences derived therefrom and degenerate variants thereof are described in the following paragraphs. However, the principles described herein include, for example, aveC homologous genes derived from other Streptomyces species, including, among others, Streptomyces hygroscopicus and Streptomyces griseochromomoneses. , Similarly applicable to other polynucleotide molecules.
[41] 5.1. Polynucleotide molecule encoding Streptomyces Avermitilis AveC gene product
[42] The present invention includes a complete aveC ORF of Streptomyces avermitilis, or a substantial portion thereof, and lacks the following complete ORF located downstream from the aveC ORF original position of the Streptomyces avermitilis chromosome. Provided are polynucleotide molecules.
[43] The isolated polynucleotide molecule of the invention is preferably identical to the Streptomyces avermitilis AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or to the nucleotide sequence of the ORF of FIG. 1 (SEQ ID NO: 1). Or a nucleotide sequence identical to a substantial portion thereof. As used herein, the “substantial portion” of an isolated polynucleotide molecule comprising a nucleotide sequence encoding a Streptomyces avermitilis AveC gene product is the complete aveC ORF sequence shown in FIG. 1 (SEQ ID NO: An isolated polynucleotide molecule comprising at least about 70% of 1) and functionally encoding an equivalent AveC gene product. Herein, the "functionally equivalent" AveC gene product is intrinsic to the Streptomyces avermitilis strain ATCC 53692 when the native aveC allele is expressed in the inactivated Streptomyces avermitilis strain ATCC 53692. It is defined as a gene product that results in producing proportions and amounts of substantially identical avermectins produced by Streptomyces avermitilis strain ATCC 53692 expressing only the wild type functional aveC allele of.
[44] In addition to the nucleotide sequence of the aveC ORF, the isolated nucleotide molecule of the present invention is a nucleotide sequence that is naturally contiguous at the original position of the aveC gene of Streptomyces avermitilis , such as the adjacent nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1). It may further comprise.
[45] The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence of SEQ ID NO: 1 or a degenerate variant thereof.
[46] As used herein, the terms “polynucleotide molecule”, “polynucleotide sequence”, “coding sequence”, “open-reading frame” and “ORF” may be single stranded or double stranded and Transcribed into the AveC gene product, or the AveC homologous product as described below, or in the same peptide as the AveC gene product or AveC homologous gene product in a suitable host cell expression system placed under the control of appropriate regulatory elements (DNA), Or both DNA and RNA molecules that can be translated (RNA). Coding sequences may include, but are not limited to, prokaryotic sequences, cDNA sequences, genomic DNA sequences, and chemically synthesized DNA and RNA sequences.
[47] The nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) comprises four different GTG codons at 42, 174, 177 and 180 bp positions. As described in paragraph 9 below, multiple deletions in the 5 ′ region of aveC ORF (FIG. 1; SEQ ID NO: 1) help to identify which of these codons can act in the aveC ORF as an initiation site for protein expression. It is comprised so that it may become. The first GTG deficiency at 42 bp did not eliminate AveC activity. Additional deletions of all GTG codons together at positions 174, 177 and 180 bp indicate a region required for protein expression as it eliminates AveC activity. Therefore, the present invention includes aveC ORFs of various lengths.
[48] The present invention is identical to the Streptomyces Abermitis AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or to the nucleotide sequence of the aveC ORF present in FIG. 1 (SEQ ID NO: 1) or a substantial portion thereof. Further provided are polynucleotide molecules having a nucleotide sequence. When used to refer to the same polynucleotide molecule as the Streptomyces avermitilis AveC gene product-coding sequence, the term "identical"
[49] (a) encodes an AveC gene product identical to the Streptomyces Abermitis AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604), or the nucleotide sequence of aveC ORF present in FIG. 1 (SEQ ID NO: 1) A nucleotide sequence that encodes the same AveC gene product but that comprises one or more silent changes to the nucleotide sequence following degeneracy (ie, degenerate variants) of the genetic code; or
[50] (b) encodes an amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or with somewhat stringent conditions (ie, 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS) and 1 mM EDTA). Hybridized to filter-bound DNA at 65 ° C. and washed in 0.2 × SSC / 0.1% SDS at 42 ° C. (Ausubel et al. (Eds.), Current Protocols in Molecular Biology, Vol. I, p.2.10.3 , 1989, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York)), the complement of a polynucleotide molecule having a nucleotide sequence encoding the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2) By a polynucleotide molecule hybridized to the host sequence and having a nucleotide sequence encoding a functionally equivalent AveC gene product as defined above. In a preferred embodiment, the homologous polynucleotide molecule is a complementary sequence of the AveC gene product-encoding nucleotide sequence of plasmid pSE186 (ATCC 209604), or 65 ° C. in very stringent conditions (ie, 0.5 M NaHPO ₄ , 7% SDS and 1 mM EDTA). Nucleotides of aveC ORF which are hybridized to filter-bound DNA in and washed in 0.1 × SSC / 0.1% SDS at 68 ° C. (see above in Ausubel et al., Supra ) in FIG. 1 (SEQ ID NO: 1). Or to a complementary sequence of a substantial portion thereof substantially, and functionally encoding the equivalent AveC gene product as defined above.
[51] The activity of the AveC gene product and its potential functional equivalents can be measured via HPLC analysis of fermentation products as described in the Examples below. Polynucleotide molecules having a nucleotide sequence encoding a functional equivalent of the Streptomyces avermitilis AveC gene product can be applied to the naturally occurring aveC gene, another Streptomyces species present in other strains of Streptomyces avermitilis . AveC homologs present, and mutated aveC alleles that are naturally occurring or genetically produced.
[52] The present invention is a polypeptide having the same amino acid sequence as that encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or having the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) or a substantial part thereof. Further provided are polynucleotide molecules comprising a nucleotide sequence that encodes. As used herein, “substantial portion” of the amino acid sequence (SEQ ID NO: 2) of FIG. 1 includes at least about 70% of the amino acid sequence (SEQ ID NO: 2) shown in FIG. 1, as defined above. Likewise refers to a polypeptide constituting a functionally equivalent AveC gene product.
[53] As used herein to refer to the same amino acid sequence as the amino acid sequence of the AveC gene product of Streptomyces avermitilis, the term "identical" has the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2), but one or more amino acid residues. Is conservatively substituted with other amino acid residues, wherein the AveC gene product of plasmid pSE186 (ATCC 209604) when said amino acid sequence is determined by all standard amino acid sequencing algorithms such as the BLASTP algorithm (NCBI from GENBANK) A poly having at least about 70%, more preferably at least about 80% and most preferably at least about 90% homology to the polypeptide encoded by the coding sequence or amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) Peptides, conservative substitutions as defined above result in functionally equivalent gene products . Conservative amino acid substitutions are well known in the art. The provisions for such substitutions are described, inter alia, in Dayhof, MD, Nat. Biomed. Res. Found ., Vol . 5 , Sup. 3, 1978, Washington, DC. More specifically, conservative amino acid substitutions generally occur within amino acid families that are related to acidity or polarity. Genetically encoded amino acids generally include (1) acidic: aspartate and glutamate; (2) basic: lysine, arginine and histidine; (3) nonpolar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; And (4) uncharged polarity: Glycine, asparagine, glutamine, cysteine, serine, threonine or tyrosine. Phenylalanine, tryptophan and tyrosine are also classified together as aromatic amino acids. One or more substitutions in any particular group, eg, leucine and isoleucine or valine, substitution of aspartate and glutamate, substitution of threonine and serine, or amino acid residues that are structurally related to any other amino acid residue, such as similar Substitution of amino acid residues with similarities in acidity or polarity, or some combination thereof, will have a meaningless effect on the function of the polypeptide.
[54] The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homologous gene product. As used herein, “AveC homologous gene product” is defined by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) as measured by all standard amino acid sequence identification algorithms such as the BLASTP algorithm (GenBank NCBI). It is defined as a gene product having at least about 50% amino acid sequence homology to the AveC gene product of Streptomyces avermitilis comprising an encoded amino acid sequence or an amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). . In a non-limiting embodiment, the AveC homologous gene product is derived from Streptomyces hygroscopicus (described in European Patent No. EP 0298423; deposited as FERM BP-1901), and the amino acid sequence of SEQ ID NO: 4 or a substantial portion thereof Include. By “substantially a portion” of the amino acid sequence of SEQ ID NO: 4 is meant a polypeptide comprising at least about 70% of the amino acid sequence of SEQ ID NO: 4 and constituting a functionally equivalent AveC homologous gene product. A "functionally equivalent" AveC homologous product was expressed in Streptomyces hygroscopicus strain FERM BP-1901 when the original aveC homolog allele was expressed in inactivated Streptomyces hygroscopicus strain FERM BP-1901. It is defined as a gene product that substantially produces the same percentage and amount of milbamycin produced by Streptomyces hygroscopicus strain FERM BP-1901 that expresses only the native wild-type functional aveC homologous alleles. In a non-limiting embodiment, the isolated polynucleotide molecules of the present invention encoding Streptomyces hygroscopius AveC homologous gene product comprise the nucleotide sequence of SEQ ID NO: 3 or a substantial portion thereof. Wherein the “substantial portion” of the isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 3 comprises at least about 70% of the nucleotide sequence of SEQ ID NO: 3 and is the functionally equivalent AveC defined immediately above. By isolated polynucleotide molecules that encode homologous gene products.
[55] The present invention further provides a polynucleotide molecule comprising a nucleotide sequence identical to the Streptomyces hygroscopicus nucleotide sequence of SEQ ID NO: 3. When used to refer to a polynucleotide molecule comprising the same nucleotide sequence as the Streptomyces hygroscopius AveC homologous gene product-encoding sequence of SEQ ID NO: 3, the term "identical"
[56] (a) a nucleotide sequence that encodes the same gene product as the nucleotide sequence of SEQ ID NO: 3 but which comprises one or more silent changes to the nucleotide sequence following degeneration of the genetic code (ie, degenerate variant); or
[57] (b) hybridize to filter-bound DNA at 65 ° C. in somewhat stringent conditions (ie, 0.5 M NaHPO ₄ , 7% SDS and 1 mM EDTA, and wash in 0.2 × SSC / 0.1% SDS at 42 ° C. Nucleotide sequence that hybridizes to the complementary sequence of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4, and that functionally encodes an equivalent AveC homologous gene product, as defined above). It means a polynucleotide molecule having. In a preferred embodiment, the homologous polynucleotide molecule is hybridized to filter-bound DNA at 65 ° C. in very stringent conditions (ie, 0.5 M NaHPO ₄ , 7% SDS and 1 mM EDTA, and at 0.1 × SSC / 0.1% SDS at 68 ° C.). Under washing (see supra et al., Supra), it hybridizes to the complementary sequence of the AveC homologous gene product-encoding nucleotide sequence of SEQ ID NO: 3 and encodes a functionally equivalent AveC homologous gene product as defined above.
[58] The present invention further provides a polynucleotide molecule comprising a nucleotide sequence encoding the same polypeptide as the Streptomyces hygroscopius AveC homologous gene product. As used herein to refer to the same polypeptide as the AveC homologous gene product of SEQ ID NO: 4 derived from Streptomyces hygroscopicus, the term "identical" has the amino acid sequence of SEQ ID NO: 4, but not more than one amino acid. The residues are conservatively substituted with other amino acid residues and, as defined above, are present in the polypeptide of SEQ ID NO: 4 when the amino acid sequence is measured by all standard amino acid sequencing algorithms such as the BLASTP algorithm (GenBank NCBI). A polypeptide having an amino acid identity of at least about 70%, more preferably at least about 80%, most preferably at least about 90%, relative to the functionally equivalent AveC homology as defined above, as defined above. Cause gene products.
[59] The present invention provides a polynucleotide molecule having a nucleotide sequence of FIG. 1 (SEQ ID NO: 1) or SEQ ID NO: 3, or a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of FIG. 1 (SEQ ID NO: 1) or SEQ ID NO: 3 Further provided are oligonucleotides that hybridize to the nucleotide molecule. The oligonucleotides are at least about 10 nucleotides in length, preferably from about 15 to about 30, and in very stringent conditions (i.e., about 14-base oligos in 6xSSC / 0.5% sodium pyrophosphate, at about 37 ° C, about 17-bases). The oligos hybridize at about 48 ° C. and about 20-base oligos at about 55 ° C.) to one of the polynucleotides). In a preferred embodiment, the oligonucleotide is complementary to a portion of one of the polynucleotide molecules. The oligonucleotides serve a variety of purposes, including acting as antisense molecules or as antisense molecules useful in gene regulation, or as primers in the amplification of polynucleotide molecules encoding AveC homologous gene products. useful.
[60] Additional aveC homologous genes can be identified in other species or strains of Streptomyces using polynucleotide molecules or oligonucleotides disclosed herein in conjunction with known techniques. For example, an oligonucleotide molecule comprising a portion of the Streptomyces avermitilis nucleotide sequence (SEQ ID NO: 1) of FIG. 1 or a portion of the Streptomyces hygroscopicus nucleotide sequence of SEQ ID NO: 3 may be detected. Labeled genomic aggregates consisting of DNA derived from the organism of interest. The stringency of the hybridization conditions is selected based on the relationship of the reference organism, for example Streptomyces avermitilis or Streptomyces hygroscopicus, with the organism of interest. The requirements for various stringent conditions are well known to those skilled in the art, and these conditions will vary as expected depending on the specific organism from which the aggregate and the labeled sequence are derived. The oligonucleotides are preferably at least about 15 nucleotides in length and include, for example, those described in the Examples below. Amplification of homologous genes may be accomplished by applying standard techniques such as polymerase chain reaction (PCR), although other amplification techniques known in the art may also be used, for example ligase chain reactions. Oligonucleotides can be used.
[61] Clones identified as containing aveC homologous nucleotide sequences can be tested for their ability to encode functional AveC homologous gene products. To this end, the clones may be necessary for sequence analysis to identify the appropriate reading frame as well as the initiation and termination signals. Alternatively or additionally, the cloned DNA sequence can be inserted into an appropriate expression vector, ie, a vector comprising elements necessary for the transcription and translation of the inserted protein-coding sequence. Any of a variety of host / vector systems can be used in bacterial systems, such as, but not limited to, plasmid, bacteriophage or cosmid expression vectors, as described below. Then, suitable host cells transformed with the vector comprising the potential aveC homologous gene coding sequence are subjected to AveC-type activity using methods such as, for example, HPLC analysis of fermentation products as described in paragraph 7 below. Can be analyzed.
[62] The production and manipulation of the polynucleotide molecules disclosed herein are known to those skilled in the art and are described, for example, in Manatis, et al., Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel, et al., Current Protocols In Molecular Biology, 1989, Greene Publishing Associations & Wiley Interscience, NY, Sambrook, et al., Molecular Cloning, A Laboratory Manual, 1989, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Innis, et al. (Eds), PCR Strategies, 1995, Academic Press, Inc., San Diego, and Erlich (ed), PCR Technology, 1992 , Oxford University Press, New York, all of which are hereby incorporated by reference. Polynucleotide clones encoding AveC gene products or AveC homologous gene products can be identified using any technique known in the art, including but not limited to, the methods described in paragraph 7 below. Genomic DNA aggregates are described for the bacteriophage aggregates (Benton and Davis, Science , 196 , 180, 1977), and for plasmid aggregates, see Grunstein and Hogness, Proc. Natl. Acad. Sci. USA , 72 , 3961-3965, 1975 can be screened for aveC gene and aveC homologous gene-coding sequences using techniques such as those described. For example, polynucleotide molecules with nucleotide sequences known to contain aveC ORFs as shown in plasmid pSE186 (ATCC 209604) or plasmid pSE119 (described in paragraph 7 below) can be used as probes in screening experiments. Alternatively, oligonucleotide probes corresponding to nucleotide sequences deduced from some or complete amino acid sequences of purified AveC homologous gene products may be synthesized.
[63] 5.2. Recombination system
[64] 5.2.1. Cloning and Expression Vectors
[65] The invention further provides recombinant cloning vectors and expression vectors useful for cloning or expressing the polynucleotide molecules of the invention, including for example aveC ORF or aveC homologous ORF of Streptomyces avermitilis . In a non-limiting embodiment, the present invention provides plasmid pSE186 (ATCC 209604) comprising the complete ORF of the aveC gene of Streptomyces avermitilis.
[66] Also, a polynucleotide molecule comprising aveC ORF derived from Streptomyces avermitilis , aveC ORF derived from Streptomyces avermitilis , or a portion thereof, or Streptomyces avermitilis AveC gene product. All of the following specification below refers to aveC homologues and AveC homologous gene products unless expressly specified in the context.
[67] A variety of different vectors have been developed for specific use in Streptomyces, including phages, high copy number plasmids, low copy number plasmids and E. coli -streptomyces shuttle vectors, among others. All of these may be used to practice the present invention. In addition, numerous drug-resistant genes have been cloned from Streptomyces and some of these genes have been incorporated into the vectors as selectable markers. Examples of conventional vectors for use in Streptomyces are described, inter alia, in Hutchinson, Applied Biochem. Biotech. , 16 , 169-190, 1980.
[68] Recombinant vectors, particularly expression vectors of the invention, are preferably configured such that the coding sequence for the polynucleotide molecule of the invention is effectively associated with one or more regulatory elements necessary to transcribe and copy the coding sequence that produces the polypeptide. As used herein, the term “regulatory element” refers to inducible and non-inducible promoters, enhancers, operators that help induce and / or regulate the expression of polynucleotide coding sequences. And nucleotide sequences encoding other elements known in the art. In addition, as used herein, the coding sequence is "effectively associated" with one or more regulatory elements where the regulatory element is effectively regulated and allowed for transcription of the coding sequence or copying of its mRNA, or both.
[69] Typical plasmid vectors that can be genetically produced to contain the polynucleotide molecules of the invention include, among others, pCR-blunt (Blunt), pCR2.1 (Invitrogen), pGEM3Zf (Promega) and Shuttle vectors pWHM3 (see Vara et al., J. Bact ., 171 , 5872-5881, 1989) are included.
[70] Methods for constructing recombinant vectors comprising specific coding sequences that are effectively associated with suitable regulatory elements are well known in the art and they can be used to demonstrate the present invention. Such methods include in vitro recombination techniques, synthetic techniques, and in vivo genetic recombination. For example, this technique is described in the literature of Maniatis et al., In the above described in Outerbell et al., In the Innis et al., And in Erlich et al.
[71] Regulatory elements of the vectors can vary in their strength and specificity. Depending on the host / vector system used, any of a number of suitable transcription and translation elements can also be used. Non-limiting examples of transcriptional regulatory regions and promoters for bacteria include β-gal promoters, T7 promoters, TAC promoters, λ left and right promoters, trp and lac promoters, trp-lac fusion promoters, and more specifically Streptomyces. Promoters ermE , melC , tipA, and the like. In a specific embodiment described in paragraph 11 below, an expression vector was generated comprising the aveC ORF cloned adjacent to a strong endurance ermE promoter derived from Saccharopolyphora erythrea . This vector was transformed into Streptomyces avermitilis , and then HPLC analysis of the fermentation product showed increased titers of avermectin produced as compared to production by the same strain expressing the wild type aveC allele.
[72] Fusion protein expression vectors can be used to express AveC gene product-fusion proteins. The purified fusion proteins can be used to increase the antiserum against AveC gene products, to study the biochemical properties of AveC gene products, to prepare AveC fusion proteins with various biochemical activities, or to express AveC gene products. It can be used to aid in identification or purification. Possible fusion protein expression vectors insert sequences encoding β-galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and polyhistidine fusions (carrier regions). Vectors include, but are not limited to. In another embodiment, the AveC gene product or portion thereof is an AveC homologous gene derived from another species or strain of Streptomyces, such as, for example, Streptomyces hygroscopicus or Streptomyces griseochromogenes. May be fused to the product or part thereof. The following paragraphs in particular embodiment described and shown in 12 Fig. 7, a chimeric plasmid containing a 564bp region of the Streptomyces Abbe reumi subtilis aveC Streptomyces high-gloss nose kusu aveC homologous ORF replacing a homologous 564bp region of the ORF Is composed. Such hybrid vectors can be tested to transform Streptomyces avermitilis, for example to determine their effect on the ratio of type 2: 1 avermectin produced.
[73] AveC fusion proteins can be made genetically to include regions useful for purification. For example, AveC-maltose-binding protein fusions can be purified using amylose resins; AveC-glutathione-S-transferase fusion proteins can be purified using glutathione-agarose beads; AveC-polyhistidine fusions can be purified using divalent nickel resins. In addition, antibodies to carrier proteins or polypeptides can be used for affinity chromatography purification of fusion proteins. For example, the nucleotide sequence encoding the target epitope of a monoclonal antibody can be genetically prepared with an expression vector that effectively associates with a regulatory element, so that the expressed antigenic determinant can be positioned to fuse to the AveC polypeptide. For example, the nucleotide sequence encoding the hydrophilic labeling peptide, FLAG, an antigenic determinant tag (International biotechnologies Inc.) can be used, for example, in the AveC polypeptide. The expression vector can be inserted by standard techniques at the point corresponding to the carboxyl terminus. The expressed AveC polypeptide-flag antigenic determinant fusion product can then be detected and affinity-purified using a commercially available anti-flag antibody.
[74] In addition, the expression vector encoding the AveC fusion protein may be a polylinker encoding a specific protease cleavage site so that the expressed AveC polypeptide may be released from a transport region or a fusion partner by treatment with a specific proteolytic enzyme. Can be prepared genetically to include the sequence. For example, the fusion protein vector may comprise a DNA sequence encoding inter alia thrombin or factor Xa cleavage site.
[75] Signal sequences upstream from the aveC ORF and with them in the reading frame can be genetically produced in expression vectors by known methods to govern the trade and secretion action of the expressed gene product. Examples of non-limiting signal sequences include those derived from α-factors, immunoglobulins, outer membrane proteins, penicillins and T-cell receptors.
[76] To aid in the selection of host cells transformed or transfected with the cloning or expression vectors of the present invention, the vectors may be genetically prepared to further comprise coding sequences for reporter gene products or other selectable labels. have. The coding sequence is preferably effectively associated with a regulatory element coding sequence as described above. Reporter genes useful in the present invention are well known in the art and include, among others, those encoding green fluorescent protein, luciferase, xylE and tyrosinase. Nucleotide sequences encoding selectable indications are well known in the art and include those that encode gene products that confer resistance to antibiotics or anti-metabolic products, or which feed nutritional requirements. Examples of such sequences include, among others, encoding resistance to erythromycin, thiostrepton or kanamycin.
[77] 5.2.2 Transformation of Host Cells
[78] The present invention further provides transformed host cells comprising the polynucleotide molecules or recombinant vectors of the invention, and novel strains or cell lines derived therefrom. Host cells useful in the practice of the invention may also be used with other prokaryotic or eukaryotic cells, although streptomyces cells are preferred. Such transformed host cells typically include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with recombinant vectors.
[79] The polynucleotide molecules of the invention function in Streptomyces cells, but can also be transformed into other bacteria or eukaryotic cells, for example for cloning or expression purposes. For example, it can be purchased from the American Type Culture Collection (ATCC; US spawn stock; Rockville, MD) (Accession No. 31343) or commercially available (Stratragene) strain DH5α. The same Escherichia coli strains can typically be used. Preferred eukaryotic host cells include yeast cells, although mammalian cells or insect cells can also be used effectively.
[80] The recombinant expression vector of the invention is preferably transformed or transfected into one or more host cells of substantially the same cell culture. The expression vector is known in the art, for example protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microscopic injection, electroporation, transfection by recombinant viral contact, liposome-mediated transfection, DEAE-dextran transfection It is common to introduce into host cells by transduction, conjugation, or microprojectile bombardment. Selection of the transformants can be performed according to standard procedures by selecting cells expressing selectable labels associated with the recombinant vector, such as antibiotic resistance, as described above.
[81] When the expression vector is introduced into a host cell, insertion and maintenance of the aveC coding sequence in the host cell chromosome or episome is performed using standard techniques such as Southern hybridization assays, restriction enzyme assays, reverse transcription enzyme PCR (rt). -PCR), or by immunological analysis to detect the expected gene product. Host cells comprising and / or expressing a recombinant aveC coding sequence include (i) a method for DNA-DNA, DNA-RNA or RNA-antisense RNA hybridization; (ii) detecting the presence of a "label" gene action; (iii) assessing the level of transcription measured by expression of aveC -specific mRNA copies in host cells; (iv) mature poly as measured by immunological assay, or by the presence of AveC biological activity (eg, the production of proportions and amounts of avermectins indicative of AveC activity in Streptomyces avermitilis host cells). It can be identified by at least four general approaches well known in the art, including methods for detecting the presence of peptide products.
[82] 5.2.3. Expression and Characterization of Recombinant AveC Gene Products
[83] When the aveC coding sequence is stably introduced into an appropriate host cell, the transformed host cell is replicated and propagated, and the resulting cells can be grown under conditions that aid in maximal production of the AveC gene product. By these conditions, cells typically grow at high density. If the expression vector contains an inducible promoter, the change in temperature, depletion of nutrients, free inducer (e.g., analogue of carbohydrates such as isopropyl-β-D-thiogalactopyranoside (IPTG)) Appropriate induction conditions such as addition, accumulation of excess metabolite byproducts, etc., are applied as necessary to induce expression.
[84] When the expressed AveC gene product is maintained inside the host cell, the cell is harvested, lysed, and at extraction conditions known in the art to minimize proteolysis, for example at 4 ° C. or in the presence of protease inhibitors. Or purify the product from the lysate under these two conditions. If the expressed AveC gene product is secreted from the host cell, the depleted nutrient medium can be simply harvested to isolate the product therefrom.
[85] The expressed AveC gene product may optionally include, but is not limited to, any combination of methods such as ammonium sulfate precipitation, size fractions, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. The method can be used to isolate or substantially purify from cell lysate or culture. If the expressed AveC gene product exhibits biological activity, increased purity of the preparation can be monitored at each step of the purification procedure using appropriate assays. Whether the expressed AveC gene product exhibits biological activity can be detected, for example, based on size or reactivity with the antibody specific for AveC or by the presence of a fusion tag. As used herein, AveC gene products consisting of at least about 20% protein by weight in certain preparations are "substantially purified". In addition, as used herein, AveC gene products consisting of at least about 80% by weight protein in certain preparations are “isolated”.
[86] Therefore, the present invention is recombinantly expressed comprising an amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) or a substantial portion thereof, and its analogs. Isolated, or substantially purified, Streptomyces avermitilis AveC gene product.
[87] The present invention further provides recombinantly expressed, isolated or substantially purified Streptomyces hygroscopicus AveC homologous gene product comprising the amino acid sequence of SEQ ID NO: 4, or a substantial portion thereof, and its analogs.
[88] The present invention includes polynucleotide molecules having a nucleotide sequence encoding the AveC gene product under conditions conducive to producing a recombinant AveC gene product and which are effectively associated with one or more regulatory elements that control the expression of the polynucleotide molecule in a host cell. Culturing the host cell transformed with the recombinant expression vector; And recovering the AveC gene product from the cell culture.
[89] Recombinantly expressed Streptomyces avermitilis AveC gene product screens compounds that modulate avermectin biosynthesis by altering the function of the AveC gene product, including increasing antibodies to the AveC gene product. Useful for
[90] If AveC gene products of sufficient purity are obtained, they can be characterized by standard methods including SDS-PAGE, size exclusion chromatography, amino acid sequencing, biological activity to produce suitable products in the avermectin biosynthetic pathway, and the like. . For example, the amino acid sequence of the AveC gene product can be measured using standard peptide sequencing techniques. The AveC gene product can be subjected to hydrophobicity and hydrophilicity analysis (see, eg, Hop and Woods, Proc. Natl. Acad. Sci. USA , 78 , 3824, 1981), or similar software algorithms. It may be further characterized to identify the hydrophilic region. Structural analysis can be performed to identify regions of the AveC gene product that are presumed to be specific secondary structures. X-ray crystallography (see Engstrom, Biochem. Expl Biol ., 11 , 7-13, 1974), computer modeling (Fletterick and Zoller (eds), Current Communications in Molecular Biology, 1973, Cold Spring Harbor Laboratory, Biophysical methods such as Cold Spring Harbor, NY), and nuclear magnetic resonance (NMR) can be used to map and study the site of interaction between the AveC gene product and its substrate. The information obtained from this study can be used to select new sites for mutations in the aveC ORF that help develop new strains of Streptomyces avermitilis with more desirable avermectin production properties.
[91] 5.3. Composition and Uses of AveC Mutant
[92] The present invention relates to the Streptomyces avermitilis aveC allele or its degenerate variant, the AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or its degenerate variant, or Streptomyces ave as present in FIG. 1. Same as the nucleotide sequence of the aveC ORF of rumitis (SEQ ID NO: 1) or its degenerate variant, but by further comprising one or more mutations, the wild type aveC allele is inactivated, resulting in inactivation of the mutated nucleotide sequence or its degenerate Avermectins different from those produced by cells of Streptomyces avermitilis strain ATCC 53692 expressing only the wild-type aveC allele expressing nucleotide molecules comprising variants Nuclei to Produce Proportions or Amounts of It provides a polynucleotide molecule comprising a sequence Tide.
[93] According to the present invention, the polynucleotide molecule can be used to produce novel identical strains that exhibit detectable changes in avermectin production compared to Streptomyces avermitilis strains expressing only the wild type aveC allele. . In a preferred embodiment, the polynucleotide molecule is useful for producing a novel identical strain that produces a reduced type 2: 1 ratio of avermectin compared to a Streptomyces avermitilis strain expressing only the wild type aveC allele. . In a further preferred embodiment, the polynucleotide molecule is useful for producing novel identical strains that produce increased levels of avermectin compared to Streptomyces avermitilis strains expressing only a single wild type aveC allele. In a further preferred embodiment, the nucleotide molecule is useful for producing novel strains of Streptomyces avermitilis in which the aveC gene is inactivated.
[94] A mutation to an aveC allele or coding sequence introduces one or more amino acid deletions, additions or substitutions into the AveC gene product, or results in cleavage of the AveC gene product or any combination thereof, producing any desired result. It includes. In addition, the mutated aveC allele sequence includes its degenerate variant. For example, the present invention relates to the nucleotide sequence of the aveC allele or its degenerate variant, the AveC gene product-coding sequence of plasmid pSE186 (ATCC 209604) or its degenerate variant, or Streptomyces ave as present in FIG. 1. Nucleotide sequence of aveC ORF of rumitis (SEQ ID NO: 1) or a degenerate variant thereof, but further comprising one or more mutations encoding a substitution of an amino acid residue for a different amino acid residue at a selected position of the AveC gene product To provide a polynucleotide molecule. In various non-limiting embodiments illustrated below, the substitutions include SEQ ID NO: 38, 48, 55, 89, 99, 111, 136, 138, 139, 154, The amino acid positions 179, 228, 230, 238, 266, 275, 289 or 298, or some combination thereof, may be performed at any amino acid position of the AveC gene product.
[95] Mutations against the aveC coding sequence are carried out by any of a variety of known methods, including the use of error-prone PCR or cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be applied to alter the sequence of aveC alleles or ORFs in a prescribed manner, for example, by introducing one or more restriction sites, or termination codons, into aveC alleles or specific regions within the ORF. Can be. In addition, methods such as those described in US Pat. Nos. 5,605,793, US Pat. Nos. 5,830,721 and 5,837,458, including random fragmentation, repeat cycles of mutation development, and nucleotide shuffling, have nucleotide sequences encoding aveC mutations. It can be used to produce huge aggregates of polynucleotides.
[96] Target mutations can be used, in particular, to change one or more conserved amino acid residues in the AveC gene product. For example, the deduced amino acid sequence of the AveC gene product as shown in Figure 6 and Streptomyces Avermitilis (SEQ ID NO: 2), Streptomyces Griseochromogenes (SEQ ID NO: 5) and By comparing the AveC homologous gene products derived from Streptomyces hygroscopicus (SEQ ID NO: 4), it is possible to indicate important conservation sites of amino acid residues between these species. Target mutations that result in changes in one or more conserved amino acid residues can be effective in producing novel mutant strains that exhibit desirable changes, particularly in avermectin production.
[97] Random mutagenesis may also be useful, and may be useful for streptophoresis in ultraviolet radiation or X-rays or chemical mutagens (eg, N-methyl-N'-nitrosoguanidine, ethyl methane sulfonate, nitrous acid or nitrogen mustard). This can be done by exposing the cells of Maises avermitilis (see, eg, Augustus's literature above for a review of mutagenesis techniques).
[98] When mutated polynucleotide molecules are produced, they are screened to determine if they can modulate the avermectin biosynthesis of Streptomyces avermitilis. In a preferred embodiment, polynucleotide molecules with mutated nucleotide sequences are tested by supplementing a Streptomyces avermitilis strain in which the aveC gene is inactivated to provide an aveC negative ( aveC ⁻ ) background. In a non-limiting embodiment, the mutated polynucleotide molecule is effectively associated with one or more regulatory elements and is preferably conjugated to an expression plasmid comprising one or more drug resistant genes for selecting transformed cells. Such vectors are then transformed into aveC ^- host cells using known techniques, and the transformed cells are selected and cultured in appropriate fermentation medium under conditions that allow or induce avermectin production. The fermentation product is then analyzed by HPLC to determine the ability of the mutated polynucleotide molecules to replenish host cells. Some vectors with mutated polynucleotide molecules capable of reducing the B2: B1 ratio of avermectin, including pSE188, pSE199, pSE231, pSE239 and pSE290 to pSE297, are illustrated in paragraph 8.3 below.
[99] The present invention provides a method for identifying mutations in aveC ORF of Streptomyces avermitilis that can alter the proportion and / or amount of avermectin produced. In a preferred embodiment, the present invention provides a method for treating a Streptomyces avermitilis strain wherein (a) the native aveC allele is inactivated so that a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product is introduced and expressed. Measuring the type 2: 1 ratio of avermectin produced by the cell; (b) the same Streptomyces avermi as in step (a) except expressing only the wild type aveC allele or aveC allele having the ORF nucleotide sequence (SEQ ID NO: 1) or the same nucleotide sequence in FIG. Measuring the type 2: 1 ratio of avermectin produced by the cells of the tilis strain; And (c) a type 2: 1 ratio of avermectin produced by Streptomyces avermitilis cells in step (a) and Aber produced by Streptomyces avermitilis cells in step (b) By comparing the type 2: 1 ratio of mectin, the type 2: 1 ratio of avermectin produced by the Streptomyces avermitilis cells of step (a) is reduced to the Streptomyces avermitilis of step (b). Identifying a mutation in the aveC ORF that can alter the type 2: 1 ratio of avermectin if it differs from the type 2: 1 ratio of avermectin produced by the cells. Provides a method for identifying mutations in aveC ORF that can change the type 2: 1 ratio. In a preferred embodiment, the type 2: 1 ratio of avermectin is reduced by mutation.
[100] In a further preferred embodiment, the present invention comprises (a) a nucleotide sequence that encodes a mutated AveC gene product, wherein the native aveC allele is inactivated, or comprises a nucleotide sequence that encodes an AveC gene product or comprises an AveC gene product. Measuring the amount of avermectin produced by the cells of a Streptomyces avermitilis strain in which a polynucleotide molecule comprising a genetic construct comprising a nucleotide sequence encoding is introduced and expressed; (b) measuring the amount of avermectin produced by the cells of the same Streptomyces avermitilis strain as in step (a), except that only the wild type aveC allele or the same nucleotide sequence is expressed ; And (c) comparing the amount of avermectin produced by the Streptomyces avermitilis cells of step (a) with the amount of avermectin produced by the Streptomyces avermitilis cells of step (b) Whereby the amount of avermectin produced by the Streptomyces avermitilis cells of step (a) is different from the amount of avermectin produced by the Streptomyces avermitilis cells of step (b), Oh mutations aveC ORF or a genetic makeup that contain that can change the amount of Verbier Abamectin aveC ORF or a step to check the mutation of the gene configuration, aveC ORF capable of changing the amount of HABERE Abamectin produced Provide a method to verify. In a preferred embodiment, the amount of avermectin is increased by mutation.
[101] Any method for identifying such mutations is preferably performed using a fermentation medium supplemented with cyclohexane carboxylic acid, although other suitable fatty acid precursors, such as any one of the fatty acid precursors shown in Table 1, may also be used.
[102] If a mutated polynucleotide molecule that regulates avermectin production in the desired direction is identified, the location of the mutation in the nucleotide sequence can be measured. For example, a polynucleotide molecule having a nucleotide sequence encoding a mutated AveC gene product can be isolated by PCR and subjected to DNA sequence analysis using known methods. By comparing the DNA sequence of the mutated aveC allele with the wild type aveC allele, the mutation (s) responsible for the change in avermectin production can be determined. As a specific non-limiting embodiment of the present invention, a single amino acid residue substitution at any residue of No. 55 (S55F), No. 138 (S138T), No. 139 (A139T) or No. 230 (G230D), or No. 138 (S138T) And the Streptomyces Avermitilis AveC gene product comprising a double substitution at position 139 (A139T or A139F) causes a change in AveC gene product function such that the proportion of type 2: 1 avermectin produced is changed. (See paragraph 8 below), wherein the amino acid positions described correspond to those present in FIG. 1 (SEQ ID NO: 2). In addition, (1) D48E / A89T; (2) S138T / A139T / G179S; (3) Q38P / L136P / E238D; (4) F99S / S138T / A139T / G179S; (5) A139T / M228T; (6) G111V / P289L; And (7) seven combinations of mutations of A139T / K154E / Q298H have been shown to effectively reduce the type 2: 1 ratio of avermectins, respectively. As used herein, such a name, such as A139T, refers to the original amino acid residue in a single letter naming, in this example alanine (A), at the designated position in this example at position 139 of the polypeptide (SEQ ID NO: 2). ), Followed by amino acid residues replacing the original amino acid residues, in this example threonine (T). Therefore, 38, 48, 55, 89, 99, 111, 136, 138, 139, 154, 179, 228, 230, 238, 266, 275, A polynucleotide molecule having a nucleotide sequence encoding a mutated Streptomyces avermitilis AveC gene product comprising amino acid substitutions or deletions at one or more amino acid positions 289 or 298 (see FIG. 1), or any combination thereof Is encompassed by the present invention.
[103] In a preferred embodiment, the mutation is
[104] (a) amino acid residue Q at position 38 replaced by P or an amino acid that is a conservative substituent for P;
[105] (b) E or amino acid residue D at position 48 replaced by an amino acid that is a conservative substituent for E;
[106] (c) amino acid residue A at position 89 replaced by T or an amino acid that is a conservative substituent for T;
[107] (d) amino acid residue F at position 99 replaced by S or an amino acid that is a conservative substituent for S;
[108] (e) amino acid residue G at position 111 replaced by V, or amino acid that is a conservative substituent on V;
[109] (f) P or amino acid residue L at position 136 replaced by amino acid that is a conservative substituent on P;
[110] (g) T, or amino acid residue S at position 138 replaced by amino acid that is a conservative substituent for T;
[111] (h) amino acid residue A at position 139 replaced by T or F or an amino acid that is a conservative substituent for T or F;
[112] (i) E or amino acid residue K at position 154 replaced by amino acid that is a conservative substituent for E;
[113] (j) amino acid residue G at position 179 replaced by S or an amino acid that is a conservative substituent for S;
[114] (k) amino acid residue M at position 228 replaced by T or an amino acid that is a conservative substituent for T;
[115] (l) amino acid residue E at position 238 replaced by D or the amino acid that is a conservative substituent for D;
[116] (m) L, or amino acid residue P at position 289 replaced by amino acid that is a conservative substituent for L; And
[117] (n) encodes an amino acid substituent selected from one or more groups consisting of amino acid residues Q at position 298 replaced by H, or an amino acid that is a conservative substituent for H, wherein the conservative amino acid substituents are as defined above in paragraph 5.1. same.
[118] In a further preferred embodiment, said mutations encode a combination of amino acid substituents and the combination of substituted amino acid residues comprises (a) amino acid residues S138 and A139; (b) amino acid residues D48 and A89; (c) amino acid residues S138, A139 and G179; (d) amino acid residues Q38, L136 and E238; (e) amino acid residues F99, S138, A139 and G179; (f) amino acid residues A139 and M228; (g) amino acid residues G111 and P289; And (h) amino acid residues A139, K154 and Q298.
[119] In a further preferred embodiment, certain combinations of mutations in the aveC allele useful for effectively reducing the type 2: 1 ratio of avermectins according to the invention include: (a) S138T / A139T; (b) S138T / A139F; (c) D48E / A89T; (d) S138T / A139T / G179S; (e) Q38P / L136P / E238D; (f) F99S / S138T / A139T / G179S; (g) A139T / M228T; (h) G111V / P289L; And (i) A139T / K154E / Q298H.
[120] The present invention further provides a composition for preparing a novel strain of Streptomyces avermitilis comprising a mutated aveC allele causing a change in avermectin production. For example, the present invention relates to any polynucleotide comprising a mutated nucleotide sequence of the present invention with respect to a site of the aveC gene of the Streptomyces avermitilis chromosome for insertion into or replacing aveC ORF or a portion thereof by homologous recombination. Provided are recombinant vectors that can be used to target nucleotide molecules. However, according to the present invention, a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention also provided herein is inserted into the Streptomyces avermitilis chromosome at a site other than the aveC gene, or the Streptomyces avermitili It also acts to regulate avermectin biosynthesis when maintained in episomes in human cells. Therefore, the present invention also includes a mutated nucleotide sequence of the present invention, which may be used to insert a polynucleotide molecule at a site of the Streptomyces avermitilis chromosome other than the aveC gene, or which may be maintained in an episome. Provided are vectors comprising polynucleotide molecules.
[121] In a preferred embodiment, the present invention provides a modified type 2 compared to cells of the same strain expressing only the wild type aveC allele by inserting the mutated aveC allele or its degenerate variant into cells of the Streptomyces avermitilis strain. Provided are gene replacement vectors that can be used to produce novel strains of Streptomyces avermitilis cells that produce one ratio of avermectin. In a preferred embodiment, the type 2: 1 ratio of avermectin produced by said cells is reduced. Such gene replacement vectors may be constructed using mutated polynucleotide molecules present in expression vectors provided herein, such as, for example, pSE188, pSE199 and pSE231, illustrated in paragraph 8 below.
[122] In a further preferred embodiment, the invention inserts a mutated aveC allele or a degenerate variant thereof into a cell of a Streptomyces avermitilis strain, thereby altering the compared to cells of the same strain expressing only the wild type aveC allele. Provided are vectors that can be used to produce novel strains of cells that produce amounts of vermectin. In a preferred embodiment, the amount of Avermecti produced by said cells is increased. In a specific but non-limiting embodiment, the vector is located upstream from the aveC allele and is effectively associated therewith, such as is known in the art as a strong endurance ermE promoter derived from Saccharophora erythrea. Additionally contains strong promoters. The vector may be plasmid pSE189 described in Example 6 below, or may be constructed using the mutated aveC allele of plasmid pSE489.
[123] In a further preferred embodiment, the present invention provides gene replacement vectors useful for inactivating aveC genes of wild-type strains of Streptomyces avermitilis . In a non-limiting embodiment, the gene replacement vector can be constructed using mutated polynucleotide molecules present in plasmid pSE180 (ATCC 209605) (FIG. 3) illustrated in paragraph 8.1 below. The present invention includes or consists of a nucleotide sequence that is naturally contiguous at the original position of the aveC gene on the Streptomyces avermitilis chromosome, including, for example, adjacent to the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1). Further provided are gene replacement vectors that can be used to delete Streptomyces avermitilis aveC ORF, including polynucleotide molecules.
[124] The present invention includes cells expressing the aveC allele and producing altered rates and / or amounts of avermectin compared to cells of the same strain of Streptomyces avermitilis expressing only the mutated wild type aveC allele. It further provides a method for preparing a novel strain of Streptomyces avermitilis. In a preferred embodiment, the invention provides a type 2 of avermectin produced by cells of the same strain expressing the mutated aveC allele compared to cells of the Streptomyces avermitilis strain expressing only the wild type aveC allele. Transforming the cells of the Streptomyces avermitilis strain with a vector having a mutated aveC allele encoding a gene product that changes one ratio; And the type-2 production by the strain expressing only the wild-type aveC allele cells:, wild-type aveC comprising the step of selecting transformed cells that produce HABERE Abamectin of: 1 ratio compared to the first rate changes the type 2 Streptomyces ave comprising cells expressing the mutated aveC allele and producing a type 2: 1 ratio of altered avermectin compared to cells of the same strain of Streptomyces avermitilis expressing only the allele Provided are methods for preparing novel strains of lumitilis. In a more preferred embodiment, the invention transforms the cells of the Streptomyces avermitilis strain with a vector capable of introducing a mutation into the aveC allele of said cell, and as a result of the mutation for the aveC allele, SEQ ID NO: 2 Of 38, 48, 55, 89, 99, 111, 136, 138, 139, 154, 179, 228, 230, 238, 266, 275, 289 By causing a substitution in the encoded AveC gene product of the different amino acid residues at one or more amino acid positions corresponding to amino acid residues at position 2 or 298, the cells of the Streptomyces avermitilis strain mutated with the aveC allele resulted in the wild type aveC allele. Streptomyces avermitilis, comprising producing a type 2: 1 ratio of avermectin that is different from the rate produced by cells of the same Streptomyces avermitilis expressing Gulf It provides a method for producing a novel strain of. In a preferred embodiment, the altered type 2: 1 ratio of avermectin is reduced.
[125] As used herein, an amino acid residue encoded by the Streptomyces avermitilis chromosome, or the aveC allele in a vector or isolated polynucleotide molecule of the present invention “corresponds to a specific amino acid residue of SEQ ID NO: 2. Or amino acid substitution occurs at a particular position “corresponding to” the specific numbered amino acid residue of SEQ ID NO: 2, which skilled artisan Reference is made to amino acid residues at the same relative positions in the AveC gene product, which can be measured quickly by reference.
[126] The present invention discloses a novel mutation wherein a specific mutation in an aveC allele that encodes a particular mutation is described as a base change at a specific nucleotide position of the aveC allele that "corresponds to" a particular nucleotide position as shown in SEQ ID NO: 1. Further provided are methods of making the strains. As noted above with respect to the corresponding amino acid position, the nucleotide position in the aveC allele is referred to as "corresponding to" the specific nucleotide position in SEQ ID NO: 1, which is the skilled person's presence of a nucleotide present herein as SEQ ID NO: 1. Reference is made to the nucleotides at the same relative positions of the aveC nucleotide sequence, which can be quickly determined by reference to the sequences.
[127] In a further preferred embodiment, the invention is directed to a subtype produced by a cell of a Streptomyces avermitilis strain expressing a mutated aveC allele or gene construct compared to cells of the same strain expressing only a single wild type aveC allele. Transforming the cells of the Streptomyces avermitilis strain with a vector having a gene construct comprising a mutated aveC allele or aveC allele causing a change in vermectin amount; And selecting the transformed cells producing a changed amount of avermectin compared to the amount of avermectin produced by the cells of the strain expressing only a single wild-type aveC allele. Provided is a method for preparing a novel strain of Streptomyces avermitilis comprising cells that produce. In a preferred embodiment, the amount of avermectin produced in the transformed cells is increased.
[128] In a further preferred embodiment, the present invention comprises the steps of transforming cells of a Streptomyces avermitilis strain expressing any aveC allele with a vector inactivating the aveC allele; And selecting transformed cells in which the aveC allele has been inactivated, thereby providing a method for preparing a novel strain of Streptomyces avermitilis comprising the inactivated aveC allele. In a preferred but non-limiting embodiment, cells of the Streptomyces avermitilis strain are transfected with a gene replacement vector having an aveC allele inactivated by mutation or by replacement of a portion of the aveC allele with a heterologous gene sequence. conversion is, the native aveC allele has been replaced with the inactivated aveC allele transformed cells are selected. Inactivation of aveC alleles can be measured by HPLC analysis of fermentation products, as described below. In a specific but non-limiting embodiment described in paragraph 8.1 below, the aveC allele is inactivated by inserting the ermE gene from Saccharopolyphora erythrea into the aveC ORF.
[129] The present invention further provides novel strains of Streptomyces avermitilis comprising cells transformed with any polynucleotide molecule or vector of the present invention. In preferred embodiments, the invention is a comprising a cell expressing the aveC allele or a degeneracy variants of the place of the wild-type aveC allele or else also mutated, produced by the same strain which expresses only the wild-type aveC allele cells Provided are novel strains of Streptomyces avermitilis that produce altered type 2: 1 ratios of avermectins as compared to the type 2: 1 ratio of avermectins. In a preferred embodiment, the altered type 2: 1 ratio produced by the novel cells is reduced. The novel strains are useful for large scale production of commercially desirable avermectins such as doramectin. In a more preferred embodiment, the invention comprises any of said mutations or combinations of mutations in the aveC allele at the nucleotide positions corresponding to those present herein above, or strepto-coding any of said amino acid substituents in the AveC gene product. Provides cells of the mises avermitilis. While the mutation may be present in the cell on extrachromosomal elements such as plasmids, it is preferred that the mutation is present in the aveC allele located on the Streptomyces avermitilis chromosome. In a preferred embodiment, the present invention comprises SEQ ID NO: 38, 48, 55, 89, 99, 111, 136, 138, 139, 154, 179, 228, 230 , Having the mutation of the aveC allele encoding the AveC gene product having a substituent at one or more amino acid positions corresponding to amino acid residues 238, 266, 275, 289 or 298, and which express the wild type aveC allele Provided are Streptomyces avermitilis strains comprising cells that produce an avermectin type 2: 1 ratio that is different from the rate produced by cells of a Streptomyces avermitilis strain.
[130] The primary purpose of the screening assays described herein is to identify mutated alleles of the aveC gene whose expression changes the ratio of type 2: 1 avermectin produced in Streptomyces avermitilis cells, and more particularly decreases. . In a preferred embodiment, the ratio of B2: B1 avermectin produced by cells of the novel Streptomyces avermitilis strain of the present invention expressing the mutated aveC allele of the present invention, or a degenerate variant thereof, is about 1.6: 1 or less. In a more preferred embodiment, this ratio is about 1: 1 or less. In a more preferred embodiment, this ratio is about 0.84: 1 or less. In a more preferred embodiment, the ratio is about 0.80: 1 or less. In a more preferred embodiment, this ratio is about 0.75: 1 or less. In a more preferred embodiment, this ratio is about 0.73: 1 or less. In a more preferred embodiment, the ratio is about 0.68: 1 or less. In an even more preferred embodiment, the ratio is about 0.67: 1 or less. In a more preferred embodiment, this ratio is about 0.57: 1 or less. In even more preferred embodiments, the ratio is about 0.53: 1 or less. In an even more preferred embodiment, the ratio is about 0.42: 1 or less. In an even more preferred embodiment, the ratio is 0.40: 1 or less.
[131] In the specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of less than 1.6: 1. In another specific embodiment described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.94: 1. In still other specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.88: 1. In still other specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.84: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.75: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.73: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.68: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin in a ratio of about 0.67: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.57: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin in a ratio of about 0.53: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin in a ratio of about 0.42: 1. In even more specific embodiments described below, the novel cells of the present invention produce cyclohexyl B2: cyclohexyl B1 avermectin at a ratio of about 0.40: 1.
[132] In a further preferred embodiment of the invention expressing the genetic composition comprising a wild-type aveC a instead of allele or else again mutated aveC allele or a degeneracy variants, or aveC allele or a degeneracy variants, the wild-type aveC Provided are the same novel strains comprising cells that produce altered amounts of avermectin compared to cells of a Streptomyces avermitilis strain that expresses only the allele. In a preferred embodiment, the novel strain produces an increased amount of avermectin. In a non-limiting embodiment, the construction of the gene further comprises a strong promoter, such as a strong endurance ermE promoter derived from Saccharofolia eritrea , located upstream from the aveC ORF and effectively related thereto.
[133] In a further preferred embodiment, the invention provides a novel strain of Streptomyces avermitilis comprising cells in which the aveC gene has been inactivated. Such strains are useful for both the spectrum of different avermectins they produce compared to wild type strains, and the supplemental screening assays disclosed herein to determine whether target or random mutagenesis of the aveC gene affects avermectin production. Do. In the specific embodiments described below, Streptomyces avermitilis host cells were genetically engineered to contain inactivated aveC genes. For example, strain SE180-11 described in the Examples below is a gene replacement plasmid pSE180 (ATCC209605) configured to inactivate Streptomyces avermitilis aveC gene by inserting an ermE resistant gene into the aveC coding region (FIG. 3). Produced).
[134] The invention further provides a mutated Streptomyces avermitilis AveC gene product, recombinantly expressed by any of the above polynucleotide molecules of the invention, and a method of making the same.
[135] The present invention changes the type 2: 1 ratio of avermectin produced by cells of the same strain expressing the mutated aveC allele compared to cells of the Streptomyces avermitilis strain expressing only the wild type aveC allele. Culturing the cells of the Streptomyces avermitilis strain expressing the mutated aveC allele encoding the gene product to be subjected to or inducing medium for production of avermectin; And recovering the avermectin from the culture, further providing a method for producing avermectin. In a preferred embodiment, the type 2: 1 ratio of avermectin produced in culture by cells expressing the mutated aveC allele is reduced. The method provides increased efficiency in producing commercially expensive avermectins such as doramectin.
[136] The present invention cells of the same strain expressing cost as compared with that only the wild-type aveC allele expressed without expression of the aveC allele or a genetic mutation configured Streptomyces Abbe reumi subtilis strain cells mutated aveC allele or genetic makeup Production of avermectin cells of a Streptomyces avermitilis strain expressing a mutated aveC allele or gene construct comprising the aveC allele resulting in producing an altered amount of avermectin produced by Culturing in medium under these acceptable or induced conditions; And recovering the avermectin from the culture, further providing a method for producing avermectin. In a preferred embodiment, the amount of avermectin produced in the culture is increased by cells expressing the mutated aveC allele, degenerate variant or gene construct.
[137] The present invention relates to a type 2 of avermectin produced by cells of the same strain expressing a mutated aveC allele or degenerate variant compared to cells of a Streptomyces avermitilis strain expressing only the wild type aveC allele. Streptomyces avermitilis , produced by a mutant aveC allele encoding a gene product that decreases by one ratio, or Streptomyces avermitilis expressing its degenerate variant, expressing only the wild type aveC allele Further provided is a novel composition of avermectins produced at a reduced type 2: 1 ratio, compared to a type 2: 1 ratio of avermectins produced by cells of the same strain of. Such novel avermectin compositions may be present when produced in depleted fermentation broth or harvested therefrom. The novel avermectin compositions can be partially or substantially purified from the culture by known biochemical purification techniques such as ammonium sulfate precipitation, dialysis, size fractionation, ion exchange chromatography, HPLC, and the like.
[138] 5.4. Uses of Avermectin
[139] Avermectins are highly active paratoxes with specific uses as parasiticides, ectoparasites, insecticides and tick insecticides. Avermectin compounds produced according to the process of the invention are useful for any of these purposes. For example, avermectin compounds produced according to the present invention are useful for treating a variety of diseases or conditions in humans, particularly diseases or conditions caused by parasitic infections as known in the art (eg, literature [ Ikeda and Omura, Chem. Rev. , 97 (7) , 2591-2609, 1997). More specifically, the avermectin compounds produced according to the present invention are used to treat various diseases or symptoms caused by internal parasites such as nematodes that can infect humans, livestock, pigs, sheep, poultry, horses or cattle. effective.
[140] More specifically, the avermectin compounds produced according to the present invention are effective against nematodes that infect humans as well as nematodes that infect various species of animals. The nematode is ansil Los Thomas (Ancylostoma), four Kato (Necator), roundworm (Ascaris), Strong jilroyi Deeds (Strongyloides), tree key Nella (Trichinella), moduchung (Capillaria), flatworms (Trichuris), strategic point (Enterobius), Parasites in the gastrointestinal tract, such as Dirofilaria , and parasitic worms found in the blood or other tissues or organs, such as filamentous worms, and the extracted visceral states of Stronggilloids and Trikinella .
[141] In addition, the avermectin compounds produced in accordance with the present invention are true mites, mites, teeth, fleas, black flies, biting insects, or juveniles that may affect in vitro parasite infections, for example cattle and horses. It is useful for treating arthropod rampant in mammals and birds, caused by larvae of twins.
[142] In addition, the avermectin compounds produced according to the present invention are not only pests of houses such as, for example, cockroaches, moths, carpet beetles and house flies, but also among them, spider mites, aphids, caterpillars and direct insects (eg Also useful as insecticides against insect pests of stored cereals and crops, including grasshoppers).
[143] Animals that can be treated with the avermectin compounds produced according to the invention include birds, dogs, and cats, including sheep, cattle, horses, deer, goats, pigs, poultry.
[144] The avermectin compounds produced according to the invention are administered in appropriate formulations depending on the particular use intended, the particular species of host animal to be treated, the type of parasite or insect to be treated. For use as an antiparasitic agent, the avermectin compounds produced according to the present invention can be administered orally in the form of capsules, pills, tablets or liquid potions, or poured and applied to the skin, injected or administered as grafts. have. Such formulations are prepared by conventional methods in accordance with standard veterinary practice. Thus, capsules, pills or tablets may be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier further comprising a disintegrant and / or a binder (eg, starch, lactose, talc, magnesium stearate, etc.). Can be. Solution formulations can be prepared by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent and the like. Injectable formulations may be prepared in the form of sterile solutions which may include sufficient salt to prepare a solution that is isotonic with blood and / or other substances such as glucose and the like.
[145] The formulation will vary with respect to the weight of the active ingredient depending on the species of the patient or host animal to be treated, the severity and type of infection, and the weight of the host. In general, for oral administration, a dose of about 0.001 to 10 mg of active ingredient per kg of patient or animal will be satisfactorily administered once or in divided doses for 1 to 5 days. However, based on clinical symptoms, it may be a substance that exhibits a higher or lower dosage range, for example, as determined by a physician or veterinarian or the like.
[146] Alternatively, the avermectin compounds produced according to the invention can be administered in admixture with animal feed, for which a concentrated feed additive or premix can be prepared for mixing with a general animal feed.
[147] As pesticides and for use in the treatment of pests of crops, the avermectin compounds produced according to the invention can be applied as sprays, dusts, emulsions and the like according to standard agricultural practice.
[148] Example 1
[149] Fermentation and B2: B1 Avermectin Analysis of Streptomyces Avermitilis
[150] Strains lacking both branched 2-oxo acid dehydrogenase and 5-O-methyltransferase activity do not produce avermectin unless the fermentation medium is supplemented with fatty acids. The above examples show that a wide range of B2: B1 ratios of avermectin can be obtained when biosynthesis is initiated in the presence of various fatty acids in such mutants.
[151] 6.1. Substances and Methods
[152] 20 g of Streptomyces Avermitilis ATCC 53692 starch (Nadex, Laing National); 15g of Pharmamedia (Trader's Protein, Memphis, Tenn.), 5g of Ardamine pH (Yeast Products Inc.), and 1g of calcium carbonate The whole culture prepared in the seed medium was stored at -70 ° C. The final volume was adjusted to 1 L with tap water, the pH was adjusted to 7.2, and then the medium was autoclave at 121 ° C. for 25 minutes.
[153] The preparation was inoculated into a flask containing 50 ml of the same medium using 2 ml of the thawed suspension. After 48 hours of incubation at 28 ° C. in a rotary shaker at 28 ° C. for 48 hours, 80 ml of starch, 7 g of calcium carbonate, 5 g of Pharmamedia, 1 g of dipotassium hydrogen phosphate, 1 g of magnesium sulfate, 0.6 g of glutamic acid, and iron sulfate ( II) A flask was inoculated with 50 ml of production medium consisting of 0.01 g of heptahydrate, 0.001 g of zinc sulfate, and 0.001 g of manganese sulfate. The final volume was adjusted to 1 L with tap water, the pH was adjusted to 7.2, and the medium was autoclaved at 121 ° C. for 25 minutes.
[154] Various carboxylic acid substrates (see Table 1 below) were dissolved in methanol and added to the fermentation broth at a final concentration of 0.2 g / l 24 hours after inoculation. After fermentation broth was incubated at 28 ° C. for 14 days, the culture was centrifuged (2 min at 2500 rpm) and the supernatant was discarded. The cell pellet was extracted with acetone (15 mL) and then dichloromethane (30 mL), the organic phase was separated, filtered and evaporated to dryness. The residue was dissolved in methanol (1 mL) and analyzed by HPLC with Hewlett-Packard 1090A liquid chromatography equipped with a scanning diode-array detector set to 240 nm. The column used was a Beckman Ultrasphere C-18, 5 μm, 4.6 mm × 25 cm column maintained at 40 ° C. 25 μl of the methanol solution was injected into the column. Elution with a linear gradient of methanol-water from 80:20 to 95: 5 over 40 minutes at a flow rate of 0.85 ml / min. Two standard concentrations of cyclohexyl B1 were used to calibrate the detector response and to measure the area under the B2 and B1 avermectin curves.
[155] 6.2. result
[156] The HPLC retention times observed for B2 and B1 avermectins, and the ratio of these avermectins 2: 1, are shown in Table 1 below.
[157] HPLC retention time (minutes)ratio temperamentB2B1B2: B1 4-tetrahydropyran carboxylic acid8.114.50.25 Isobutyric acid10.818.90.5 3-furic acid7.614.60.62 S-(+)-2-methylbutyric acid12.821.61.0 Cyclohexane carboxylic acid16.926.01.6 3-thiophene carboxylic acid8.816.01.8 Cyclopentane carboxylic acid14.223.02.0 3-trifluoromethylbutyric acid10.918.83.9 2-methylpentanoic acid14.524.94.2 Cycloheptane carboxylic acid18.629.015.0
[158] The data present in Table 1 show a very wide B2: B1 avermectin product ratio, which varies considerably depending on the nature of the fatty acid side chain initiator unit given the result of dehydration of the type 2 compound to the type 1 compound. Indicates that there is. This means that changes in the B2: B1 ratio due to modification of the AveC protein may be specific for a particular substrate. As a result, screening for mutants showing changes in the B2: B1 ratio obtained with a particular substrate needs to be done in the presence of the substrate. Subsequent examples described below use cyclohexane carboxylic acid as the screening substrate. However, these substrates are merely used to illustrate the possibilities and do not limit the applicability of the present invention.
[159] Example 2
[160] aveCIsolation of genes
[161] This example describes the isolation and characterization of the Streptomyces Avermitilis chromosomal region encoding the AveC gene product. As described below, it was confirmed that the aveC gene can change the ratio of cyclohexyl-B2: cyclohexyl-B1 (B2: B1) of avermectin produced.
[162] 7.1. Substances and Methods
[163] 7.1.1. Culture of Streptomyces for DNA Isolation
[164] The following method was involved for culturing Streptomyces. A single colony (single colony isolate # 2) of Streptomyces Avermitilis ATCC 31272 was prepared using 5 g of Difco yeast extract, 5 g of Difco Bacto-peptone, 2.5 g of dextrose, 5 g of MOPS and difco. It was isolated at 1/2 concentration YPD-6 containing 15 g of bacto-agar. The final volume was adjusted to 1 L with distilled water, the pH was adjusted to 7.0 and the medium was autoclaved at 121 ° C. for 25 minutes.
[165] TSB medium (1 L autoclaved for 25 minutes at 121 ° C.) in a 25 mm × 150 mm tube maintained with shaking (300 rpm) at 28 ° C. for 48 to 72 hours using the cells grown on the medium. 10 ml of difcotrypsin digested soybean culture in distilled water) was inoculated.
[166] 7.1.2. Chromosomal DNA Isolation from Streptomyces
[167] Aliquots (0.25 ml or 0.5 ml) of the cells grown as described above were placed in a 1.5 ml micro centrifuge tube and the cells were concentrated by centrifugation at 12,000 xg for 60 seconds. Discard the supernatant and separate cells with 0.25 mL of TSE buffer containing 2 mg / mL of lysozyme (20 mL of 1.5M sucrose, 2.5 mL of 1M Tris-HCl, pH 8.0, 2.5 mL of 1M EDTA, pH 8.0 and 75 mL of distilled water). Resuspended in the air. The samples were incubated at 37 ° C. for 20 minutes with shaking, and then placed on an AutoGen 540® automated nucleic acid isolation device (Integrated Separation Systems, Natick, Mass.). The genomic DNA was packed and isolated using Cycle 159 (device software) according to the manufacturer's instructions.
[168] Alternatively, 5 ml of cells were placed in a tube of 17 mm x 100 mm, the cells were concentrated by centrifugation at 3,000 rpm for 5 minutes, and then the supernatant was removed. The cells were resuspended in 1 ml of TSE buffer, concentrated by centrifugation at 3,000 rpm for 5 minutes, and then the supernatant was removed. The cells were resuspended in 1 ml TSE buffer containing 2 mg / ml lysozyme and incubated at 37 ° C. with shaking for 30-60 minutes. After incubation, 0.5 ml of 10% sodium dodecyl sulfate (SDS) was added and incubated at 37 ° C. until the cells were completely lysed. The lysates were incubated at 65 ° C. for 10 minutes, cooled to room temperature, then divided into two 1.5 ml Eppendorf tubes and pre-equilibrated with 0.5 ml of phenol / chloroform (0.5 M Tris, pH 8.0). 50% phenol; 50% chloroform). The aqueous phase was removed and extracted 2-5 times with chloroform: isoamyl alcohol (24: 1). The DNA was precipitated by adding 1/10 volume of 3M sodium acetate (pH 4.8), the mixture was incubated for 10 minutes on ice, then centrifuged at 5 ° C. for 10 minutes at 15,000 rpm and the supernatant was emptied. 1 volume of isopropanol was added to the tube. The supernatant and isopropanol mixture was then incubated on ice for 20 minutes, centrifuged at 5 ° C. for 20 minutes at 15,000 rpm, then the supernatant was removed and the DNA pellet was washed once with 70% ethanol. After drying the pellet, the DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
[169] 7.1.3. Isolation of Plasmid DNA from Streptomyces
[170] An aliquot of the cells (1.0 mL) was placed in a 1.5 mL microcentrifuge tube and the cells concentrated by centrifugation at 12,000 xg for 60 seconds. The supernatant was discarded, the cells were resuspended in 1.0 ml of 10.3% sucrose, concentrated by centrifugation at 12,000 xg for 60 seconds, and the supernatant was discarded. Cells were then resuspended in 0.25 ml of TSE buffer containing 2 mg / ml lysozyme, incubated for 20 minutes at 37 ° C. with shaking, and then charged into an Autogen 540 automated nucleic acid separation device. Plasmid DNA was isolated using Cycle 106 (device software) according to the manufacturer's instructions.
[171] Alternatively, 1.5 ml of cells were placed in a 1.5 ml micro centrifuge tube and the cells were concentrated by centrifugation at 12,000 xg for 60 seconds. The supernatant was discarded, the cells were resuspended in 1.0 ml of 10.3% sucrose, concentrated by centrifugation at 12,000 xg for 60 seconds, and the supernatant was discarded. The cells were resuspended in 0.5 ml TSE buffer containing 2 mg / ml lysozyme and incubated at 37 ° C. for 15-30 minutes. After incubation, 0.25 ml of alkali SDS (0.3 N NaOH, 2% SDS) was added and the cells were incubated at 55 ° C. for 15-30 minutes or until the solution was clear. Sodium acetate (0.1 ml, 3M, pH 4.8) was added to the DNA solution and then incubated on ice for 10 minutes. The DNA sample was centrifuged at 14,000 prm at 5 ° C. for 10 minutes. The supernatant was removed and 0.2 mL of phenol: chloroform (50% phenol; 50% chloroform) was added to the emptied tube and mixed slowly. The DNA solution was centrifuged at 14,000 rpm at 5 ° C. for 10 minutes and the top layer was removed to empty the Eppendorf tubes. Isopropanol (0.75 mL) was added and the solution was slowly mixed and then incubated for 20 minutes at room temperature. The DNA solution was centrifuged at 5 ° C. for 15 minutes at 14,000 rpm, then the supernatant was removed and the DNA pellet was washed with 70% ethanol and dried and then resuspended in TE buffer.
[172] 7.1.4. Isolation of Plasmid DNA from Escherichia coli
[173] Transformed Escherichia coli single colonies were mixed with 5 ml of Luria-Bertani (LB) medium (10 g of bacto-tryptone, 5 g of bacto-yeast extract and 10 g of NaCl in 1 L of distilled water for 25 minutes at 121 ° C. Autoclave and supplemented with 100 μg / ml ampicillin). Cultures were incubated overnight and 1 ml aliquots were placed in 1.5 ml micro centrifuge tubes. Culture samples were filled in an Autogen 540 automated nucleic acid isolation device and plasmid DNA was isolated using Cycle 3 (device software) according to the manufacturer's instructions.
[174] 7.1.5. Preparation and Transformation of Streptomyces Avermitilis Protoplasts
[175] Single colonies of Streptomyces avermitilis were isolated at 1/2 concentration YPD-6. The cells were inoculated into 10 ml of TSB medium in a 25 mm x 150 mm tube and then incubated for 48 hours at 28 ° C with shaking (300 rpm). 1 ml of cells were used to inoculate 50 ml of YEME medium. The YEME medium contains 3 g of difco yeast extract, 5 g of difco bacto-peptone, 3 g of difco malt extract, and 300 g of sucrose per liter. After autoclaving at 121 ° C. for 25 minutes, ₂ ml of 2.5M MgCl ₂ .6H ₂ O (separately autoclaved at 121 ° C. for 25 minutes) and 25 ml of glycine (20%) (filter-sterilized) were added. .
[176] The cells were grown at 30 ° C. for 48-72 hours and harvested by centrifugation for 20 minutes at 3,000 rpm in a 50 ml centrifuge tube (Falcon). Discard the supernatant, sucrose 205 g, K ₂ SO ₄ 0.25 g, MgCl ₂ · 6H ₂ O 2.02 g, H ₂ O 600 mL, K ₂ PO ₄ (0.5%) 10 mL, trace element solution (1 element for trace element solution) ZnCl ₂ 40 mg, FeCl ₃ · 6H ₂ O 200 mg, CuCl ₂ · 2H ₂ O 10 mg, MnCl ₂ · 4H ₂ O 10 mg, Na ₂ B ₄ O ₇ · 10H ₂ O 10 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O 10 mg) in a P buffer containing 20 mL), 100 mL of CaCl ₂ · 2H ₂ O (3.68%), and 10 mL of MES buffer (1.0M, pH 6.5). The cells were resuspended. After adjusting the pH to 6.5 and the final volume to 1 L, the medium was filtered through a 0.45 μm filter at high temperature.
[177] The cells were pelleted at 3,000 rpm for 20 minutes, the supernatant was discarded and the cells were resuspended in 20 ml P buffer containing 2 mg / ml lysozyme. The cells were incubated at 35 ° C. for 15 minutes while shaking, and observed under a microscope to determine the degree of protoplast formation. When protoplast formation was complete, the protoplasts were centrifuged at 8,000 rpm for 10 minutes. The supernatant was removed and the protoplasts resuspended in 10 ml P buffer. The protoplasts were centrifuged at 8,000 rpm for 10 minutes, the supernatant was removed, and then resuspended in 2 ml of P buffer to distribute about 1 × 10 ⁹ protoplasts into 2.0 ml cryogenic vials.
[178] Vials containing 1 × 10 ⁹ protoplasts were centrifuged at 8,000 rpm for 10 minutes, the supernatant was discarded, and the protoplasts were resuspended in 0.1 ml P buffer. 2-5 μg of DNA to be transformed was added to the protoplasts, followed immediately by 0.5 ml of reaction T buffer. T buffer base solution contains 25 g PEG-1000 (Sigma), 2.5 g sucrose and 83 ml H ₂ O. The pH was adjusted to 8.8 with 1N NaOH (filter-sterilized) and the T buffer base solution was filter-sterilized and then stored at 4 ° C. Reaction T buffer is prepared and used as the same and contains 8.3 ml of T buffer base solution, 1.0 ml of ₄ mM K ₂ PO ₄ , 0.2 ml of 5M CaCl ₂ · 2H ₂ O and 0.5 ml of 1M TES (pH 8). Each component of reaction T buffer was separately filter-sterilized.
[179] Within 20 seconds of adding T buffer to the protoplasts, 1.0 mL of P buffer was also added, and then the protoplasts were centrifuged at 8,000 rpm for 10 minutes. The supernatant was discarded and the protoplasts resuspended in 0.1 ml P buffer. The protoplasts were then sucrose 205 g, K ₂ SO ₄ 0.25 g, MgCl ₂ .6H ₂ O 10.12 g, glucose 10 g, diphco casamino acid 0.1 g, diphco-yeast extract 5 g, diphco-oatmeal agar, 3 g deep Plated on RM14 medium containing 22 g co-bacto agar, and 800 ml distilled water. This solution was autoclaved at 121 ° C. for 25 minutes. After autoclaving, 10 ml of 0.5% K ₂ PO ₄ , 5 ml of 5M CaCl ₂ .2H ₂ O, 15 ml of 20% L-proline, 10 ml of 1.0M MES buffer (pH 6.5), trace element solution (as described above) Sterile stock of 2 ml, cycloheximide stock (25 mg / ml) and 2 ml of 1N NaOH were added. 25 ml of RM14 medium per plate was divided and dried for 24 hours prior to use of this plate.
[180] Protoplasts were incubated at 95% humidity and 30 ° C. for 20-24 hours. To select thiostrepton resistant transformants, 1 ml of overlay buffer containing 125 μg / ml thiostrepton was uniformly applied on RM14 regeneration plates. Overfp buffer contains 10.3 g sucrose per 100 ml, 0.2 ml trace element solution (as described above) and 1 ml 1M MES pH 6.5. The protoplasts were incubated at 95% humidity and 30 ° C. for 7-14 days until thiostrepton resistant (Thio®) colonies were found.
[181] 7.1.6. Streptomyces lividans ( Streptomyces lividansProtoplast Transformation
[182] Streptomyces lividans TK64 (provided by John Innes Institute, Norwich, UK) was used for transformation in some cases. Methods and compositions for culturing, protoplasting and transformation of Streptomyces lividans are described in Hopwood et al., Genetic Manipulation of Streptomyces , A Laboratory Manual, John Innes Foundation, Norwich, UK, 1985, It was carried out as described in the above document. Plasmid DNA was isolated from Streptomyces lividans transformants as described in Example 7.1.3 above.
[183] 7.1.7. Fermentation Analysis of Streptomyces Avermitilis Strains
[184] Streptomyces avermitilis cells grown for 4-7 days on 1/2 concentration of YPD-6 were inoculated into a 1 × 6 inch tube containing 8 ml of pre-prepared medium and two 5 mm glass beads. . Pre-prepared medium contains 20 g / l of soluble starch (lightly boiled starch, or KOSO (Japan Corn Starch Co., Nagoya, Japan)); Pharmamedia 15 g / l; Ardamine pH (Champlain Ind., Clifton, NJ) 5 g / l; CaCO ₃ 2 g / l; And 2x bcfa ("bcfa" refers to branched chain fatty acids) comprising a final concentration in a medium of 50 ppm 2 (+/-)-methylbutyric acid, 60 ppm isobutyric acid and 20 ppm isovaleric acid. The pH was adjusted to 7.2 and the medium autoclaved at 121 ° C. for 25 minutes.
[185] The tube was shaken at 29 ° C. for 3 days at 215 rpm with an angle of 17 °. 160 g / l of starch (lightly boiled starch or KOSO) using 2 ml aliquots of seed culture, Nutrisoy (Archer Daniels Midland, Decatur, Ill.) l, ardamine pH 10 g / l, K ₂ HPO ₄ 2 g / l, MgSO ₄ 4H ₂ O 2 g / l, FeSO ₄ · 7H ₂ O 0.02 g / l, MnCl ₂ 0.002 g / l, ZnSO ₄ Production medium comprising 0.002 g / l 7H ₂ O, 14 g / l CaCO ₃ , 2x bcfa (as defined above) and 800 ppm cyclohexane carboxylic acid (CHC) (prepared in 20% solution at pH 7.0) 25 A 300 ml Erlenmeyer flask containing ml was inoculated. The pH was adjusted to 6.9 and the medium autoclaved at 121 ° C. for 25 minutes.
[186] After inoculation, the flasks were incubated at 29 ° C. for 12 days with shaking at 200 rpm. After incubation, 2 ml of the sample was recovered from the flask, diluted with 8 ml of methanol and mixed, and then the mixture was centrifuged at 1,250 xg for 10 minutes to pellet the remainder. The supernatant was then analyzed by HPLC using a Beckman Ultrasphere ODS column (25 cm x 4.6 mm inner diameter) at a flow rate of 0.75 ml / min and absorbance measured at 240 nm. Mobile phase was 86 / 8.9 / 5.1 methanol / water / acetonitrile.
[187] 7.1.8. Isolation of Streptomyces Avermitilis PKS Gene
[188] Ketocinases prepared from cosmid syntheses of streptomyces avermitilis (ATCC 31272, SC-2) chromosomal DNA and prepared from fragments of Saccharopolyoratria polyketide synthase (PKS) gene (KS) hybridized with probe. A detailed description of the process for preparing cosmid aggregates can be found in Sambrook et al., Supra. A detailed description of the preparation of Streptomyces chromosomal DNA aggregates is provided in Hopwood et al., Supra. Cosmid clones containing the ketocinase-hybridization region were identified by hybridizing to a 2.7 Kb Nde I / Eco 47III fragment from pEX26 (kindly provided to Dr. P. Leadlay, Cambridge, UK). About 5 ng of pEX26 was digested with Nde I and Eco 47III. The reaction mixture was packed on 0.8% SeaPlaque GTG agarose gel (FMC BioProducts, Rockland, Maine, USA). After electrophoresis, a 2.7 Kb Nde I / Eco 47III fragment was excised from the gel and the gel was obtained using GELase (trademark) purchased from Epicentre Technologies using the Fast protocol. DNA was recovered from. 2.7 Kb Nde I / Eco 47III Fragments were manufactured using the BRL Nick Translation System (BRL Life Technologies, Inc., Gaithersburg, Md.) [Α- ³² P] dCTP (deoxycytidine 5'-triphosphate, tetra (triethylammonium) salt, [α- ³² P]-) according to the company's instructions (NEN-Dupont, Boston, MA) Dupont)). Typical reactions are carried out in a volume of 0.05 ml. After addition of 5 μl of termination buffer, the labeled DNA was not incorporated into the nucleotides according to the manufacturer's instructions using a G-25 Sephadex Quick Spin (Behringer Mannheim) column. Separated from.
[189] About 1,800 cosmid clones were screened by colony hybridization. Ten clones were identified that hybridized strongly to Saccharopolyfora Eritrea KS probe. Escherichia coli colonies containing cosmid DNA were grown in LB liquid medium and cosmid DNA from each culture was used in cycle 3 (device software) using the manufacturer's instructions on an Autogen 540 automated nucleic acid isolation device. Isolated. Restriction endonuclease map and Southern blot hybridization analysis revealed that the five clones contained overlapping chromosomal regions. The Streptomyces Abermitis genome Bam HI restriction map of five cosmids (ie pSE65, pSE66, pSE67, PSE68 and pSE69) consisted of overlapping and hybridization analysis of cosmids (FIG. 4).
[190] 7.1.9. Identification of DNA regulating the ratio of avermectin B2: B1, and aveCConfirmation of the ORF
[191] The following method was used to test the ability of subcloned fragments derived from pSE66 cosmid clones to modulate the ratio of avermectin B2: B1 in the AveC mutant strain. pSE66 (5 μg) was digested with Sac I and Bam HI. The reaction mixture was packed on 0.8% Seaplaque GTG agarose gel (FMC Bioproducts), and after electrophoresis, 2.9 Kb of Sac I / Bam HI fragments were excised from the gel and then gelled using a fast protocol ( Epicenter Technologies) was used to recover DNA from the gel. About 5 μg of the shuttle vector pWHM3 (see Vara et al., J. Bacteriol ., 171 , 5872-5881, 1989) was digested with Sac I and Bam HI. Approximately 0.5 μg of 2.9 Kb insert and 0.5 μg of digested pWHM3 are mixed together and 1 unit of ligase (New England Biolabs, Beverly, Mass., USA) in a total volume of 20 μl. Biolabs, Inc.) was incubated overnight at 15 ° C. according to the manufacturer's instructions. After incubation, 5 μl of the ligation mixture was incubated for 10 minutes at 70 ° C., cooled to room temperature, and then used to prepare the Competent Escherichia coli DH5α cells (BRL) according to the manufacturer's instructions. Used to transform. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of a 2.9 Kb Sac I / Bam HI insert was confirmed by restriction analysis. The plasmid was named pSE119.
[192] Protoplasts of Streptomyces avermitilis strain 1100-SC38 (Pfizer in-house strain) were prepared and transformed with pSE119 as described in paragraph 7.1.5. Above. Strain 1100-SC38 is a mutant strain that produces more avermectin cyclohexyl B2 form when supplemented with cyclohexane carboxylic acid (a ratio of B2: B1 is about 30: 1). PSE119, which was used to transform Streptomyces avermitilis protoplasts, was used by Dr. B. J. Bach, director of the Escherichia coli strain GM2163 (general stock center of the Escherichia coli Genetic Stock Center, Yale University, USA). Bay (obtained from BJ Bachmann), Escherichia coli strain DM1 (BRL) or Streptomyces lividans strain TK64. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and fermentation products analyzed by HPLC analysis. Transformants of Streptomyces avermitilis strain 1100-SC38 comprising pSE119 produced a changed ratio of Avermectin cyclohexyl B2: cyclohexyl B1 of about 3.7: 1 (see Table 2 below).
[193] As pSE119 demonstrated that it could regulate the avermectin B2: B1 ratio in the AveC mutant, the sequence of the inserted DNA was analyzed. About 10 μg of pSE119 was isolated using a plasmid DNA isolation kit (Qiagen, Valencia, Calif.) According to the manufacturer's instructions, and an ABI 373A automated DNA sequence analyzer (Perkin, Foster City, Calif.) The sequence was analyzed using Elkin (Perkin Elmer). Sequence data were combined and edited using the Genetic Computer Group program (GCG, Madison, Wisconsin). DNA sequence and aveC ORF are shown in FIG. 1 (SEQ ID NO: 1).
[194] A new plasmid named pSE118 was constructed as follows. About 5 μg of pSE66 was digested with Sph I and Bam HI. The reaction mixture was packed on 0.8% Seaplaque GTG agarose gel (FMC Bioproducts), and after electrophoresis, the 2.8 Kb Sph I / Bam HI fragment was excised from the gel and then gelase (Epi) using Fast protocol. Center Technologies) was used to recover DNA from the gel. About 5 μg shuttle vector pWHM3 was digested with Sph I and Bam HI. About 0.5 μg of 2.8 Kb insert and 0.5 μg of digested pWHM3 were mixed together and incubated overnight at 15 ° C. using 1 unit of ligase (New England Biolabs) in a total volume of 20 μl, according to the manufacturer's instructions. . After incubation, 5 μl of the ligation mixture was incubated for 10 minutes at 70 ° C., cooled to room temperature, and then Compident Escherichia coli DH5α cells were used for transformation according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of a 2.8 Kb Sph I / Bam HI insert was confirmed by restriction analysis. The plasmid was named pSE118. The insert DNA of pSE118 and pSE119 was overlapped with about 838 nucleotides (see FIG. 4).
[195] Protoplasts of Streptomyces avermitilis strain 1100-SC38 were transformed with pSE118 as described above. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and fermentation products analyzed by HPLC analysis. Transformants of Streptomyces avermitilis strain 1100-SC38 comprising pSE118 did not change the ratio of Avermectin cyclohexyl B2: Avermectin cyclohexyl B1 compared to strain 1100-SC38 (Table 2 below). Reference).
[196] 7.1.10. From Streptomyces Avermitilis Chromosome DNA aveCPCR amplification of genes
[197] About 1.2 Kb fragment comprising aveC ORF was isolated from Streptomyces avermitilis chromosomal DNA by PCR amplification using primers designed based on the aveC nucleotide sequence obtained above. PCR primers were purchased from Genosys Biotechnologies, Inc., Texas, USA. The right primer was 5'-TCACGAAACCGGACACAC-3 '(SEQ ID NO: 6) and the left primer was 5'-CATGATCGCTGAACCGAG-3' (SEQ ID NO: 7). The PCR reaction was performed using a Deep Vent polymerase (New England Biolabs) in a buffer provided by the manufacturer, with a final volume of 100 μl, 300 μM dNTP, 10% glycerol, 200 pmol of each primer, template 0.1 It was performed using a Perkin-Elmer Setus thermal cycler in the presence of μg and 2.5 units of enzyme. The thermal profile of the first cycle was 95 ° C. (denature step) for 5 minutes, 60 ° C. (annealing step) for 2 minutes and 72 ° C. (stretching step) for 2 minutes. Subsequent 24 cycles had similar thermal profiles except shortening the denaturation step to 45 seconds and shortening the annealing step to 1 minute.
[198] The PCR product was electrophoresed on a 1% agarose gel and a single DNA band of about 1.2 Kb was detected. The DNA was purified from the gel and linked to 25 ng of the linearized blunt pCR-blunt vector (Invitrogen) in a molar ratio of 1:10 vector: insert according to the manufacturer's instructions. Using this ligation mixture, One Shot (Company) Competent Escherichia coli cells (Invitrogen) were transformed according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of about 1.2 Kb of insert was confirmed by restriction analysis. The plasmid was named pSE179.
[199] Insertion DNA derived from pSE179 was isolated by digestion with Bam HI / Xba I, isolated by electrophoresis, purified from gel and shuttle vector pWHM3 digested with Bam HI / Xba I at a total DNA concentration of 1 μg. And the molar ratio of vector: insert of 1: 5. This ligation mixture was used to transform Competent Escherichia coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of about 1.2 Kb of insert was confirmed by restriction analysis. Plasmid DNA was isolated from ampicillin resistant transformants by transforming Escherichia coli DM1 into the plasmid designated pSE186 (FIG. 2, ATCC 209604).
[200] 7.2. result
[201] It was confirmed that the 2.9 Kb Sac I / Bam HI fragment derived from pSE119 significantly changed the ratio of B2: B1 avermectin production when transforming Streptomyces avermitilis strain 1100-SC38. Streptomyces avermitilis strain 1100-SC38 typically has a B2: B1 ratio of about 30: 1, but is about 3.7: 1 when transformed with a vector comprising a 2.9 Kb Sac I / Bam H1 fragment. Have a reduced B2: B1 avermectin ratio. Analysis of the transformed culture after fermentation confirmed the presence of the transforming DNA.
[202] The sequence of the 2.9 Kb pSE119 fragment was analyzed and an ORF of about 0.9 Kb (FIG. 1, SEQ ID NO: 1) was identified, which ORF was premutated Pst I / Sph I fragment at another location to produce only B2 product. (See the above-mentioned document by Ikeda et al.). Comparison of the ORF or its corresponding deduced polypeptide against a known database (GenEMBL, SWISS-PROT) showed no strong homology with known DNA or protein sequences.
[203] Table 2 below shows the results of fermentation analysis of Streptomyces avermitilis strain 1100-SC38 transformed with various plasmids.
[204] Streptomyces avermitilis strain (transgenic plasmid)Number of transformants testedAverage ratio of B2: B1 1100-SC38 (none)930.66 1100-SC38 (pWHM3)2131.3 1100-SC38 (pSE119)123.7 1100-SC38 (pSE118)1230.4 1100-SC38 (pSE185)1427.9
[205] Example 3
[206] Composition of Streptomyces Avermitilis AveC Mutant
[207] This example describes the construction of a variety of Streptomyces avermitilis AveC mutant strains using the compositions and methods described above. For a general description of techniques for introducing mutations in the genes of Streptomyces, see Kiers and Hopwood, Meth. Enzym. , 204, 430-458, 1991. A more detailed description can be found in Anzai et al., J. Antibiot ., XLI (2) , 226-233, 1988 and Stutzman-Engwall et al., J. Bacteriol ., 174 (1) , 144-154. , 1992. The above references are hereby incorporated by reference in their entirety.
[208] 8.1. Streptomyces Avermitilis aveC Inactivation of genes
[209] AveC mutants comprising the inactivated aveC gene were constructed using several methods as described below.
[210] In the first method, the 640 bp Sph I / Pst I fragment inside the aveC gene of pSE119 ( plasmid described in paragraph 7.1.9. Above ) was replaced with the ermE gene (erythromycin resistance) derived from Saccharophora erythrea. . The ermE gene was digested with restriction enzymes by Bgl II and Eco RI from pIJ4026 (John Inz Institute, Norwich, UK; see also Bibb et al., Gene , 41 , 357-368, 1985). It was electrophoresed and purified from the gel and isolated. This approximately 1.7 Kb fragment was linked to pGEM7Zf (Promega) digested with Bam HI and Eco RI, and this linkage mixture was transformed with Competent Escherichia coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of about 1.7 Kb inserts was confirmed by restriction analysis. The plasmid was named pSE27.
[211] pSE118 (described in paragraph 7.1.9. above) was digested with Sph I and Bam HI and the digest was electrophoresed, then about 2.8 Kb of Sph I / Bam HI inserts were purified from the gel. After pSE119 was digested with Pst I and Eco RI and the digest was electrophoresed, about 1.5 Kb of Pst I / Eco RI insert was purified from the gel. Shuttle vector pWHM3 was digested with Bam HI and Eco RI. After pSE27 was digested with Pst I and Sph I and the digest was electrophoresed, about 1.7 Kb of Pst I / Sph I insert was purified from the gel. All four fragments (ie, about 2.8 Kb, about 1.5 Kb, about 7.2 Kb, about 1.7 Kb) were all linked together in a four-way linkage. The linkage mixture was prepared by the Competent Escherichia coli DH5α cells. Transformed as directed. The plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. This plasmid was named pSE180 (FIG. 3, ATCC 209605).
[212] Streptomyces lividans TK64 was transformed with pSE180, and the transformed colonies were confirmed by resistance to thiostrepton and erythromycin. pSE180 was isolated from Streptomyces lividans and used to transform Streptomyces avermitilis protoplasts. Four thiostrepton resistant Streptomyces avermitilis transformants were identified, protoplasts prepared, and plated under non-selective conditions on RM14 medium. After regeneration of the protoplasts, single colonies with erythromycin resistance and no thiostrepton resistance were screened, indicating that the inactivated aveC gene was inserted into the chromosome and the free replicon was lost. One Erm ^r Thio ^s transformant was identified and named strain SE180-11. The whole chromosomal DNA was isolated from strain SE180-11, digested with restriction enzymes of Bam HI, Hind III, Pst I or Sph I, electrophoresed on 0.8% agarose gel and transferred to nylon membrane to ermE probe Hybridized. The analysis showed that chromosomal insertion of the ermE resistant gene and simultaneous deletion of the 640 bp Pst I / Sph I fragment occurred as a double crossing event. Analysis of the fermentation product of strain SE180-11 by HPLC showed that normal avermectin was no longer produced (FIG. 5A).
[213] As a second method for inactivating the aveC gene, 1.7 Kb of the ermE gene was removed from the chromosome of Streptomyces avermitilis strain SE180-11, resulting in a deletion of 640 bp Pst I / Sph I fragment in the aveC gene. . Gene replacement plasmids were constructed as follows: pSE180 was partially digested with Xba I and a fragment of about 11.4 Kb was purified from the gel. There was a deficiency of 1.7Kb ermE resistant gene in the about 11.4Kb band. The DNA was then linked and transformed into Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. Escherichia coli DM1 was transformed with the plasmid designated pSE184 and plasmid DNA was isolated from ampicillin resistant transformants. The plasmid was used to transform Streptomyces avermitilis strain SE180-11. Protoplasts were prepared from thiostrepton resistant transformants of strain SE180-11 and plated in single colonies on RM14. After regeneration of the protoplasts, a single colony was screened without both erythromycin resistance and thiostrepton resistance, meaning that the inactivated aveC gene was inserted into the chromosome and the free replicon containing the ermE gene was lost. . One Erm ^s Thio ^s transformant was identified and named SE184-1-13. Fermentation analysis of SE184-1-13 confirmed that SE184-1-13 did not produce normal avermectin and had the same fermentation profile as SE180-11.
[214] As a third method of inactivating the aveC gene, a Bsp E1 site was prepared by introducing a frameshift by adding 2 G below C at nucleotide 471 of the chromosome aveC gene using PCR. The presence of genetically engineered BspE1 sites was useful for detecting gene replacement events. PCR primers for introducing frame shift mutations into the aveC gene were designed and purchased from Genosys Biotechnology Inc. The right primer was 5'-GGTTCCGGATGCCGTTCTCG-3 '(SEQ ID NO: 8) and the left primer was 5'-AACTCCGGTCGACTCCCCTTC-3' (SEQ ID NO: 9). PCR conditions were as described in paragraph 7.1.10. Above. The 666 bp PCR product was digested with Sph I to give two fragments of 278 bp and 388 bp, respectively. 388 bp fragment was purified from the gel.
[215] Gene replacement plasmids were constructed as follows: Shuttle vector pWHM3 was digested with Eco RI and Bam HI. After pSE119 was digested with Bam HI and Sph I and the digest was electrophoresed, about 840 bp fragment was purified from the gel. After pSE119 was digested with Eco RI and Xmn I and the digest was separated by electrophoresis, about 1.7 Kb fragment was purified from the gel. All four fragments (ie about 7.2Kb, about 840bp, about 1.7Kb and 388bp) were all linked together in a four way link. This ligation mixture was used to transform Compitetent Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. Escherichia coli DM1 was transformed with the plasmid designated pSE185 and plasmid DNA was isolated from ampicillin resistant transformants. The plasmid was used to transform the protoplasts of Streptomyces avermitilis strain 1100-SC38. . Thiostrepton resistant transformants of strain 1100-SC38 were isolated and fermentation products analyzed by HPLC analysis. pSE185 hardly changed the B2: B1 avermectin ratio when transforming Streptomyces avermitilis strain 1100-SC38 (see Table 2).
[216] pSE185 was used to transform the protoplasts of Streptomyces avermitilis to introduce a frame shift mutation into the chromosome aveC gene. Protoplasts were prepared from thiostrepton resistant transformants and plated into single colonies on RM14. After regeneration of the protoplasts, single colonies with no thiostrepton resistance were screened. Chromosomal DNA was isolated from thiostrepton sensitive colonies and screened by PCR for the presence of a frame shift mutation inserted into the chromosome. PCR primers were designed based on the aveC nucleotide sequence and were purchased from Gynosis Biotechnology Inc. (Texas, USA). The right direction PCR primer is 5'-GCAAGGATACGGGGACTAC-3 '(SEQ ID NO: 10), the left direction PCR primer is 5'-GAACCGACCGCCTGATAC-3' (SEQ ID NO: 11), and PCR conditions are described in paragraph 7.1.10. As described. The PCR product obtained was 543 bp and three fragments of 368 bp, 96 bp and 79 bp were observed when digested with Bsp El, indicating that the inactivated aveC gene was inserted into the chromosome and the free replicon was lost.
[217] The fermentation and analysis of the Streptomyces avermitilis mutant strain containing the frame shift mutation of the aveC gene revealed that these mutants no longer produce normal avermectin, strains SE180-11 and SE 184-1-13. It was confirmed to have the same fermentation HPLC profile as. One Thio ^s transformant was identified and named strain SE185-5a.
[218] In addition, a mutant strain at the aveC gene that changed from G to A at 520 nucleotide position was produced, resulting in a change in the codon encoding tryptophan (W) at the 116 position to a stop codon. This mutated Streptomyces avermitilis strain did not produce normal avermectin and had the same fermentation profile as strains SE180-11, SE184-1-13 and SE185-5a.
[219] (I) from G to A at the nucleotide 970 position, changing the amino acid at position 256 from glycine (G) to aspartate (D); (ii) Mutants in the aveC gene were prepared which change the amino acid at position 275 from tyrosine (Y) to histidine (H), from T to C at the 996 nucleotide position. This mutated (G256D / Y275H) Streptomyces avermitilis strain did not produce normal avermectin and had the same fermentation profile as strains SE180-11, SE184-1-13 and SE185-5a.
[220] Streptomyces avermitilis aveC inactivating mutant strains SE180-11, SE184-1-13, SE185-5a and other strains provided herein provide a screening tool for assessing the effects of other mutations of the aveC gene. pSE186 comprising a wild type copy of the aveC gene transformed Escherichia coli DM1 and plasmid DNA was isolated from ampicillin resistant transformants. This pSE186 DNA was used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated and the presence of erythromycin resistance was determined, and then the fermentation products were analyzed for Thio ^r Erm ^r transformants by HPLC analysis. The presence of the trans functional aveC gene was able to recover strain SE180-11 to produce normal avermectin (see FIG. 5B).
[221] 8.2. Changing type B2: B1 ratio aveC Analysis of Mutations in Genes
[222] As described above, Streptomyces avermitilis strain SE180-11 comprising the inactive aveC gene can be supplemented by transformation with a plasmid (pSE186) comprising the functional aveC gene. In addition, strain SE180-11 was used as a host strain to characterize other mutations of the aveC gene as described below .
[223] Chromosome DNA was isolated from strain 1100-SC38 and used as a template for PCR amplification of the aveC gene. ORK of 1.2 Kb was isolated by PCR amplification using primers designed based on aveC nucleotide sequence. The right primer was SEQ ID NO: 6 and the left primer was SEQ ID NO: 7 (see paragraph 7.1.10 above). PCR and subcloning conditions are described in paragraph 7.1.10 above. Analysis of the DNA sequence of 1.2 Kb ORF revealed a mutation in the aveC gene that changes the amino acid at position 55 from serine (S) to phenylalanine (F) and changes from C to T at nucleotide position 337. Found that happened. Producing the aveC gene containing the S55F mutation named by subcloning the pWHM3 plasmid pSE187, and which was used to transform protoplasts of Streptomyces Abbe reumi subtilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and then Thio ^r Erm ^r transformants were analyzed by analyzing the fermentation products by HPLC. The presence of the aveC gene encoding the change at amino acid residue 55 (S55F) can restore strain SE180-11 to produce normal avermectin (FIG. 5C); Compared to strain SE180-11 transformed with pSE186 having a ratio of cyclohexyl B2: cyclohexyl B1 of about 1.6: 1, the ratio of B2: B1 was about 26: 1, from which the single mutation (S55F) It can be seen that it controls the amount of cyclohexyl B2 produced relative to cyclohexyl B1.
[224] Another mutation was identified in the aveC gene that changes the amino acid at position 230 from glycine (G) to aspartate (D), and from G to A at nucleotide position 862. Streptomyces avermitilis strains having this mutation (G230D) produce avermectin at a ratio of B2: B1 of about 30: 1.
[225] 8.3. Mutations that decrease the B2: B1 ratio
[226] Several mutations that reduced the amount of cyclohexyl B2 produced relative to cyclohexyl B1 were constructed as follows.
[227] Mutations in the aveC gene that change the amino acid at position 139 from alanine (A) to threonine (T), from G to A at nucleotide position 588, have been identified. To the aveC gene containing the A139T mutation into pWHM3 it subcloned producing the plasmids named as pSE188, and which was used to transform protoplasts of Streptomyces Abbe reumi subtilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and then Thio ^r Erm ^r transformants were analyzed by HPLC analysis of the fermentation products. The presence of the mutated aveC gene encoding a change in amino acid residue 139 (A139T) can restore strain SE180-11 to produce normal avermectin (FIG. 5D); The ratio of B2: B1 is about 0.94: 1, indicating that the mutation reduced the amount of cyclohexyl B2 produced relative to cyclohexyl B1. These results were not predicted as well as the results of the mutations described above, as published results indicate only inactivation of the aveC gene or increased production of the B2 form of avermectin compared to the B1 form (see Table 3 below).
[228] Since the A139T mutation changes the ratio of B2: B1 in the direction of producing more desirable B1, the mutation was constructed to encode threonine instead of serine at the amino acid position 138. Thus, pSE186 was digested with the Eco RI, was cloned into the pGEM3Zf (Promega) digested with Eco RI. This plasmid, designated pSE186a, was digested with Apa I and Kpn I, and DNA fragments were separated on agarose gels, followed by purification of two fragments of about 3.8 Kb and about 0.4 Kb from the gel. About 1.2 Kb of insert DNA from pSE186 was used as the PCR template to introduce a single base change at nucleotide position 585. PCR primers were designed to introduce mutations at the 585 nucleotide position and were purchased from Gynesis Biotechnology Inc. (Texas). The rightward PCR primer was 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3 '(SEQ ID NO: 12) and the leftward PCR primer was 5'-GGAACCGACCGCCTGATACA-3' (SEQ ID NO: 13). PCR reactions were performed using an Advantage GC Genome PCR Kit (Clonetech Laboratories, Palo Alto, Calif.) In a buffer provided by the manufacturer, 200 μM dNTP, 200 pmol of each primer, 50 ng. The final volume was performed at 50 μl in the presence of template DNA, 1.0M GC-melt and 1 unit of KlenTaq Polymerase Mix. The thermal profile of the first cycle was 94 ° C. for 1 minute, followed by 25 cycles at 94 ° C. for 30 seconds and 68 ° C. for 2 minutes, followed by one cycle at 68 ° C. for 3 minutes. 295bp PCR product is digested with Apa I and Kpn I and then release the 254bp fragment and to separate them by electrophoresis, purified from the gel. All three fragments (about 3.8 Kb, about 0.4 Kb and 254 bp) were linked together in a three way link. This ligation mixture was transformed into Competent Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. This plasmid was named pSE198.
[229] pSE198 was digested with Eco RI, cloned into pWHM3 digested with Eco RI, and then transformed into Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid DNA was transformed into Escherichia coli DM1 and the plasmid DNA was isolated from ampicillin resistant transformants, and then the presence of the correct insert was confirmed by restriction analysis. The plasmid named pSE199 was used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and then Thio ^r Erm ^r transformants were analyzed by analyzing the fermentation products by HPLC. The presence of the mutated aveC gene encoding a change in amino acid residue 138 (S138T) can restore strain SE180-11 to produce normal avermectin; The ratio of B2: B1 is about 0.88: 1, indicating that the mutation resulted in a reduction in the amount of cyclohexyl B2 produced relative to cyclohexyl B1 (see Table 3 below). The B2: B1 ratio was even lower than the ratio of 0.94: 1 observed by the A139T mutation produced in strain SE180-11 transformed with pSE188 as described above.
[230] Another mutation was constructed to introduce threonine at both amino acid positions 138 and 139. About 1.2 Kb of insert DNA obtained from pSE186 was used as a PCR template. PCR primers were designed to introduce mutations at the nucleotide positions 585 and 588 and were purchased from Gynesis Biotechnology Inc. (Texas). The right PCR primer was 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3 '(SEQ ID NO: 14), and the left PCR primer was 5'-GGAACATCACGGCATTCACC-3' (SEQ ID NO: 15). PCR reactions were performed using the conditions described earlier in this paragraph. 449bp PCR product is digested with Apa I and Kpn I and then release the 254bp fragment and to separate them by electrophoresis, purified from the gel. pSE186a is digested with Apa I and Kpn I, the DNA fragment was separated on agarose gel and purified two fragments of about 3.8Kb and about 0.4Kb from the gel. All three fragments (about 3.8 Kb, about 0.4 Kb and 254 bp) were linked together in a three-way linkage, and this linkage mixture was transformed into Competent Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. The plasmid was named pSE230.
[231] pSE230 was digested with Eco RI, cloned into pWHM3 digested with Eco RI, and then transformed into Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. The plasmid DNA was transformed into Escherichia coli DM1 and the plasmid DNA was isolated from ampicillin resistant transformants, and then the presence of the correct insert was confirmed by restriction analysis. The plasmid named pSE231 was used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. Thiostrepton resistant transformants of SE180-11 were isolated, the presence of erythromycin resistance was measured, and then Thio ^r Erm ^r transformants were analyzed by fermentation. The presence of a double mutated aveC gene encoding S138T / A139T can restore strain SE180-11 to produce normal avermectin, but with a B2: B1 ratio of about 0.84: 1, wherein the mutation is pSE188 as described above. Or for the reduction provided by strain SE180-11 transformed with pSE199, it can be seen that it further reduces the amount of cyclohexyl B2 produced relative to cyclohexyl B1 (see Table 3 below).
[232] Another mutation was further constructed to further reduce the amount of cyclohexyl B2 relative to cyclohexyl B1. Since the S138T / A139T mutant changed the B2: B1 ratio in the direction of producing more desirable B1, the mutation was constructed to introduce threonine at amino acid position 138 and phenylalanine at amino acid position 139. About 1.2 Kb of insert DNA obtained from pSE186 was used as a PCR template. PCR primers were designed to introduce mutations at the 585 nucleotide position (change from T to A), the 588 nucleotide position (change from G to T), and the 589 nucleotide position (change from C to T), and genosys It was purchased from Bio Technologies Inc. (Texas). The rightward PCR primer was 5'-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGTTC-3 '(SEQ ID NO: 25) and the leftward PCR primer was 5'-GGAACATCACGGCATTCACC-3' (SEQ ID NO: 15). PCR reactions were performed using an Advantage GC Genome PCR Kit (Clontech Laboratories, Palo Alto, Calif.) In a buffer provided by the manufacturer, using 200 μM dNTP, 200 pmol of each primer, 50 ng of template DNA, 1.1 mM Mg. The final volume was performed at 50 μl in the presence of acetate, 1.0 M GC-melt and 1 unit of Tth DNA polymerase. The thermal profile of the first cycle was 94 ° C. for 1 minute, followed by 25 cycles at 94 ° C. for 30 seconds and 68 ° C. for 2 minutes, followed by one cycle at 68 ° C. for 3 minutes. 449bp PCR product is digested with Apa I and Kpn I and then release the 254bp fragment and to separate them by electrophoresis, purified from the gel. All three fragments (about 3.8 Kb, about 0.4 Kb and 254 bp) were linked together in a three way link. This ligation mixture was transformed into Competent Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis. The plasmid was named pSE238.
[233] pSE238 was digested with Eco RI, cloned into pWHM3 digested with Eco RI, and then transformed into Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. The plasmid DNA was transformed into Escherichia coli DM1 and the plasmid DNA was isolated from ampicillin resistant transformants, and then the presence of the correct insert was confirmed by restriction analysis. This plasmid, designated pSE239, was used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and then Thio ^r Erm ^r transformants were analyzed by HPLC analysis of the fermentation products. The presence of the double mutated aveC gene encoding S138T / A139F can restore strain SE180-11 to produce normal avermectin; The B2: B1 ratio is about 0.75: 1 and the mutation is the amount of cyclohexyl B2 produced relative to cyclohexyl B1 for the reduction provided by strain SE180-11 transformed with pSE188, pSE199 or pSE231 as described above. It can be seen that it decreases (see Table 3 below).
[234] Streptomyces avermitilis strain (transformation plasmid)Number of transformants testedRelative B2 ConcentrationRelative B1 ConcentrationAverage ratio of B2: B1 SE180-11 (none)30000 SE180-11 (pWHM3)30000 SE180-11 (pSE186)262221401.59 SE180-11 (pSE187)122831126.3 SE180-11 (pSE188)241932060.94 SE180-11 (pSE199)181551710.88 SE180-11 (pSE231)62593090.84 SE180-11 (pSE239)201842420.75
[235] Stemmer, Nature , 370 , 389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. Further mutations were constructed to further reduce the amount of cyclohexyl B2 produced relative to cyclohexyl B1 using DNA shuffling techniques as described in USA , 91 , 10747-10751, 1994, US Pat. No. 5,605,793 Nos. 5,811,238, 5,830,721 and 5,837,458.
[236] DNA shuffled plasmids containing the mutated aveC gene were transformed into competent dam dcm Escherichia coli cells. Plasmid DNA was isolated from ampicillin resistant transformants and used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated and screened for production of avermectins with a cyclohexyl B2: cyclohexyl B1 ratio of less than 1: 1. The DNA sequence of the plasmid DNA obtained from the SE180-11 transformant producing avermectin with a B2: B1 ratio of 1: 1 or less was determined.
[237] Eight transformants were identified which reduced the amount of cyclohexyl B2 produced relative to cyclohexyl B1. The lowest B2: B1 ratio achieved among these transformants was 0.4: 1 (see Table 4 below). Plasmid DNA was isolated from each of the eight transformants and DNA sequences were determined to identify mutations in the aveC gene. The mutation is as follows.
[238] pSE290 comprises four nucleotide mutations changed from T to A at 317 nucleotide position, from C to A at nucleotide position 353, from G to A at 438 nucleotide position, and from T to A at nucleotide position 1155. . The nucleotide change at the nucleotide 317 position changes the amino acid from D to E at the AA position 48 and the nucleotide change at the 438 nucleotide position changes the amino acid from A to T at the AA position 89. The B2: B1 ratio produced by the cells carrying the plasmid was 0.42: 1 (see Table 4 below).
[239] pSE291 contains four nucleotide mutations changed from G to A at 272 nucleotide position, from T to A at nucleotide position 585, from G to A at nucleotide position 588, and from G to A at 708 nucleotide position. . The nucleotide change at nucleotide position 585 changes amino acid from S to T at AA position 138, the nucleotide change at nucleotide position 588 changes amino acid from A to T at AA position 139, nucleotide position 708 The nucleotide change in changes the amino acid from G to S at position 179 AA. The B2: B1 ratio produced by the cells carrying the plasmid was 0.57: 1 (see Table 4 below).
[240] pSE292 contains four nucleotide mutations identical to pSE290. The B2: B1 ratio produced by the cells carrying this plasmid was 0.40: 1 (see Table 4 below).
[241] pSE293 from A to G at nucleotide position 24, A to C at nucleotide position 286, T to C at nucleotide position 497, C to T at 554 nucleotide position, T to C at 580 nucleotide position And 6 nucleotide mutations changed from A to T at nucleotide position 886. The nucleotide change at nucleotide position 286 changes the amino acid from Q to P at the AA position 38, the nucleotide change at 580 nucleotide position changes the amino acid from L to P at the AA position 136 and the nucleotide position 886 The nucleotide change in changes the amino acid from E to D at position 238 AA. The B2: B1 ratio produced by the cells carrying the plasmid was 0.68: 1 (see Table 4 below).
[242] pSE294 from T to C at 469 nucleotide position, from T to A at nucleotide position 585, from G to A at nucleotide position 588, from G to A at nucleotide position 708, from C to T at nucleotide position 833; And 6 nucleotide mutations changed from G to A at nucleotide position 1184. In addition, the nucleotides at positions 173, 174 and 175 are deleted. The nucleotide change at 469 nucleotide position changes the amino acid from F to S at the AA position 99, the nucleotide change at nucleotide position 585 changes the amino acid from S to T at the AA position 138 and the nucleotide position 588 The nucleotide change in changes the amino acid from A to T at the AA position 139 and the nucleotide change at the 708 nucleotide position changes the amino acid from G to S at the AA position 179. The B2: B1 ratio produced by the cells carrying the plasmid was 0.53: 1 (see Table 4 below).
[243] pSE295 contains two nucleotide mutations changed from G to A at nucleotide position 588 and from T to C at 856 nucleotide position. The nucleotide change at the nucleotide position 588 changes the amino acid from A to T at the AA position 139 and the nucleotide change at the 856 nucleotide position changes the amino acid from M to T at the AA position 228. The B2: B1 ratio produced by the cells carrying the plasmid was 0.80: 1 (see Table 4 below).
[244] pSE296 from T to C at 155 nucleotide position, from G to T at 505 nucleotide position, from C to T at nucleotide position 1039, from C to T at nucleotide position 1202 and from T at nucleotide position 1210. 5 nucleotide mutations changed to C. The nucleotide change at the nucleotide position 505 changes the amino acid from G to V at the AA position 111 and the nucleotide change at the 1039 nucleotide position changes the amino acid from P to L at the AA position 289. The B2: B1 ratio produced by the cells carrying the plasmid was 0.73: 1 (see Table 4 below).
[245] pSE297 comprises four nucleotide mutations changed from G to T at nucleotide position 377, from G to A at nucleotide position 588, from A to G at 633 nucleotide position, and from A to T at nucleotide position 1067. . The nucleotide change at nucleotide position 588 changes the amino acid from A to T at AA position 139, the nucleotide change at 633 nucleotide position changes the amino acid from K to E at AA position 154, and nucleotide position 1067. The nucleotide change in changes the amino acid from Q to H at the AA position at position 298. The B2: B1 ratio produced by the cells carrying the plasmid was 0.67: 1 (see Table 4 below).
[246] Streptomyces avermitilis strain (transformation plasmid)Number of transformants testedRelative B2 ConcentrationRelative B1 ConcentrationAverage ratio of B2: B1 SE180-11 (none)4000 SE180-11 (pWHM3)4000 SE180-11 (pSE290)4872080.42 SE180-11 (pSE291)41061850.57 SE180-11 (pSE292)4912310.40 SE180-11 (pSE293)41231800.68 SE180-11 (pSE294)4681290.53 SE180-11 (pSE295)42172710.80 SE180-11 (pSE296)One1351860.73 SE180-11 (pSE297)One1482210.67
[247] Example 4
[248] 5 'deletion mutant composition
[249] As described in paragraph 5.1 above, the nucleotide sequence (SEQ ID NO: 1) of Streptomyces avermitilis shown in FIG. Four different GTG codons. This example describes the configuration of multiple deletions in the 5 'region of the aveC ORF (FIG. 1; SEQ ID NO: 1) to identify which of these codons can serve as the initiation site of the aveC ORF for protein expression.
[250] Fragments of variously deleted aveC genes at the 5 'end were isolated by PCR amplification from the chromosomal DNA of Streptomyces avermitilis . PCR primers were designed based on aveC DNA sequences and were purchased from Gynesis Biotechnology Inc. Right directional primers are 5′-AACCCATCCGAGCCGCTC-3 ′ (SEQ ID NO: 16) (D1F1); 5'-TCGGCCTGCCAACGAAC-3 '(SEQ ID NO: 17) (D1F2); 5'-CCAACGAACGTGTAGTAG-3 '(SEQ ID NO: 18) (D1F3); And 5'-TGCAGGCGTACGTGTTCAGC-3 '(SEQ ID NO: 19) (D2F2). Left primers are 5′-CATGATCGCTGAACCGA-3 ′ (SEQ ID NO: 20); 5'-CATGATCGCTGAACCGAGGA-3 '(SEQ ID NO: 21); And 5'-AGGAGTGTGGTGCGTCTGGA-3 '(SEQ ID NO: 22). PCR reactions were performed as described in paragraph 8.3 above.
[251] PCR products were separated by electrophoresis on a 1% agarose gel and a single DNA band of about 1.0 Kb or about 1.1 Kb was detected. This PCR product was purified from the gel and linked to 25 ng of the linearized pCR2.1 vector (Invitrogen) in a 1:10 mole ratio vector: insert according to the manufacturer's instructions. This linkage mixture was used to transform One Shot® Competent Escherichia coli cells (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis and DNA sequence analysis. The plasmids were named pSE190 (obtained with primers D1F1), pSE191 (obtained with primers D1F2), pSE192 (obtained with primers D1F3), and pSE193 (obtained with primers D2F2).
[252] Insert DNA was digested with Bam HI / Xba I, separated by electrophoresis, purified from gels, and then added in a 1: 5 molar ratio of 1 μg total vector: insertion to shuttle vector pWHM3 digested with Bam HI / Xba I. Linking at DNA concentration. This ligation mixture was used to transform Competent Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis. These plasmids named pSE194 (D1F1), pSE195 (D1F2), pSE196 (D1F3), and pSE197 (D2F2) were transformed into Escherichia coli strain DM1 and isolated from plasmid DNA from ampicillin resistant transformants, respectively. The presence of the correct insert was confirmed by restriction analysis. The DNA was used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. The thiog ^r Erm ^r transformant was isolated by isolating the thiostrepton resistant transformant of strain 180-11, measuring the presence of erythromycin resistance, and then analyzing the fermentation product by HPLC to determine the GTG site for aveC expression. It was confirmed that. The results showed that the GTG codon at position 42 could be eliminated without affecting aveC expression because of lack of the GTG site at position 42 but three GTG at positions 174, 177 and 180 This is because the sites including pSE194, pSE195 and pSE196 were able to recover to produce normal avermectin when transformed into SE180-11. Strain SE180-11 did not recover to produce normal avermectin when transformed with pSE197 lacking all four GTG sites (see Table 5 below).
[253] Streptomyces avermitilis strain (transformation plasmid)Number of transformants testedRelative B2 ConcentrationRelative B1 ConcentrationAverage ratio of B2: B1 SE180-11 (none)6000 SE180-11 (pWHM3)6000 SE180-11 (pSE186)62411521.58 SE180-11 (pSE194)635152.43 SE180-11 (pSE195)674381.97 SE180-11 (pSE196)63282081.58 SE180-11 (pSE197)12000
[254] Example 5
[255] From Streptomyces hygroscopius and Streptomyces griseochromogenes aveCCloning of Homologs
[256] The present invention provides a method for identifying and cloning aveC homologous genes derived from other avermectin-producing species or milbamycin -producing species of Streptomyces . For example, cosmid aggregates of genomic DNA of Streptomyces hygroscopicus (FERM BP-1901) were hybridized with a 1.2 Kb aveC probe obtained from Streptomyces avermitilis described above. Several strongly hybridized cosmid clones have been identified. Chromosomal DNA was isolated from these cosmids, and a Kpn I fragment was identified that the aveC 4.9Kb probe and hybridization. The sequence of this DNA was analyzed and an ORF (SEQ ID NO: 3) with very large homology to the aveC ORF of Streptomyces avermitilis was identified. The amino acid sequence (SEQ ID NO: 4) deduced from Streptomyces hygroscopicus aveC homologous ORF is shown in FIG. 6.
[257] In addition, cosmid syntheses of Streptomyces griseochromogenes genomic DNA were hybridized with 1.2 Kb aveC probes obtained from the Streptomyces avermitilis described above. Several strongly hybridized cosmid clones have been identified. Chromosomal DNA was isolated from these cosmids and identified a 5.4 Kb Pst I fragment that hybridized with the aveC probe. The sequence of the DNA was analyzed and the aveC homologous partial ORF with very large homology to the aveC ORF of Streptomyces avermitilis was identified. The deduced partial amino acid sequence (SEQ ID NO: 5) is shown in FIG. 6.
[258] DNA and amino acid sequences of aveC homologues obtained from Streptomyces hygroscopicus and Streptomyces griseochromogenes show that these regions are contiguous with each other and with Streptomyces avermethylis aveC ORF and AveC genes. It was found that all of the products share significant homology (about 50% sequence identity at the amino acid level) (FIG. 6).
[259] Example 6
[260] ermEUnder the promoter aveCConstruction of the plasmid with the gene
[261] a 1.2Kb aveC ORF derived from pSE186, was the shuttle vector pWHM3 having the pSE34 ermE promoter of the 300bp insert a Kpn I / Bam HI fragment in the Kpn I / Bam HI site of pWHM3 subcloning (lit. [Ward et al. , Mol. Gen. Genet ., 203 , 468-478, 1986). pSE186 was digested with Bam HI and Hind III, and this digest was isolated by electrophoresis, and then 1.2Kb fragments were isolated from agarose gels and linked to pSE34 digested with Bam HI and Hind III. This ligation mixture was transformed into Competent Escherichia coli DH5α cells according to the manufacturer's instructions. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of 1.2 Kb inserts was confirmed by restriction analysis. The plasmid designated pSE189 was transformed into Escherichia coli DM1 and plasmid DNA was isolated from ampicillin resistant transformants. pSE189 transformed the protoplasts of Streptomyces avermitilis strain 1100-SC38. Thiostrepton resistant transformants of strain 1100-SC38 were isolated and fermentation products were analyzed by HPLC analysis.
[262] The Streptomyces avermitilis strain 1100-SC38 transformant comprising pSE189 produced a ratio of avermectin cyclohexyl B2: cyclohexyl B1 (about 3: 1) compared to strain 1100-SC38 (34: 1). ), And the total production of avermectin was increased about 2.4-fold compared to strain 1100-SC38 transformed with pSE119 (see Table 6 below).
[263] In addition, pSE189 was transformed into protoplasts of wild-type Streptomyces avermitilis strains. Thiostrepton resistant transformants were isolated and fermentation products were analyzed by HPLC analysis. The total avermectin produced in wild type Streptomyces avermitilis transformed with pSE189 was increased about 2.2-fold compared to wild type Streptomyces avermitilis transformed with pSE119 (see Table 6 below).
[264] Streptomyces avermitilis (transformation plasmid)Number of transformants testedRelative B2 ConcentrationRelative B1 ConcentrationRelative Avermectin Total ProductionAverage ratio of B2: B1 1100-SC3861554.817633.9 1100-SC38 (pSE119)923950.33574.7 1100-SC38 (pSE189)165461668493.3 Wild type659421131.41 Wild type (pSE119)62481514811.64 Wild type (pSE189)55453451,0711.58
[265] Example 7
[266] Streptomyces Avermitilis aveCORF and Streptomyces hygroscopius aveCChimeric Plasmids Containing Sequences Derived from Homologs
[267] A hybrid plasmid named pSE350 was constructed as follows, comprising a portion of 564 bp of Streptomyces hygroscopicus aveC homologue that replaces a 564 bp homology portion of Streptomyces avermitilis aveC ORF. pSE350, was constructed using a Bsa AI restriction site (225-position of the aveC), and Streptomyces Abbe reumi subtilis Kpn I restriction sites present in the aveC gene (810-position in the aveC) to be stored in both sequences . PCR conditions described in paragraph 7.1.10 above, right directional primer 5'-CTTCAGGTGTACGTGTTCG-3 '(SEQ ID NO: 23) and left directional primer 5'-GAACTGGTACCAGTGCCC-3' (SEQ ID NO: 24) (purchased from Gynosis Biotechnology) The Kpn I site was introduced into Streptomyces hygroscopicus DNA by PCR. The PCR product is digested with Bsa AI and Kpn I, was isolated after separation by electrophoresis, the fragments from the 1% agarose gel, the Bsa AI / Kpn I fragment of 564bp from the gel. pSE179 (described in paragraph 7.1.10 above) was digested with Kpn I and Hind III, and fragments were separated by electrophoresis on a 1% agarose gel, then about 4.5 Kb fragment was isolated from the gel. pSE179 was digested with Hind III and Bsa AI, fragments were separated by electrophoresis on a 1% agarose gel, and then about 0.2 Kb of Bsa AI / Hind III fragment was isolated from the gel. A Streptomyces high-gloss nose kusu about a 4.5Kb Hind III / Kpn I fragment obtained from the approximately 0.2Kb the Bsa AI / Hind III fragment, and the Bsa AI / Kpn I fragments of 564bp and connected together in a three-way connection, This ligation mixture was transformed into Competent Escherichia coli DH5α cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis using Kpn I and Ava I. The plasmid was digested with Hind III and Xba I to release 1.2 Kb of insert and then linked to pWHM3 digested with Hind III and Xba I. This ligation mixture was transformed into Competent Escherichia coli DH5α cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using Hind III and Ava I. The plasmid DNA was transformed into Escherichia coli DM1 and plasmid DNA was isolated from ampicillin resistant transformants, and then the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis. This plasmid was named pSE350 and used to transform the protoplasts of Streptomyces avermitilis strain SE180-11. Thioreptone resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and the Thio ^r Erm ^r transformants were analyzed by HPLC analysis of the fermentation products. As a result, the transformants comprising the hybrid plasmid of Streptomyces avermitilis / streptomyces hygroscopicus were found to have an average B2: B1 ratio of about 109: 1 (see Table 7 below).
[268] Streptomyces avermitilis strain (transformation plasmid)Number of transformants testedRelative B2 ConcentrationRelative B1 ConcentrationAverage ratio of B2: B1 SE180-11 (none)8000 SE180-11 (pWHM3)8000 SE180-11 (pSE350)162332109
[269] Deposit of Biological Substances
[270] The following biological materials were deposited on 29 January 1998 at the American Type Culture Collection (ATCC), Rockville Parkron Drive 12301, MD, USA, and received the following accession number:
[271] PlasmidAccession number Plasmid pSE180209605 Plasmid pSE186209604
[272] All patents, patent applications and publications cited above are hereby incorporated by reference in their entirety.
[273] The specific embodiments described herein are intended to illustrate individual embodiments of the invention one by one, without limiting the scope of the invention, and functionally equivalent methods and components thereof are also included within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing specification and the accompanying drawings. Such modifications are included within the scope of the claims appended hereto.

权利要求:
Claims (81)
[1" claim-type="Currently amended] Streptomyces avermitilis aveC allele, the sequence encoding the Streptomyces avermitilis AveC gene product of plasmid pSE186 (ATCC 209604), or as present in FIG. 1 (SEQ ID NO: 1). Identical to the nucleotide sequence of the aveC ORF of Streptomyces avermitilis , or their degenerate variants, but 38, 48, 89, 99, 111, 136, 154 of SEQ ID NO: 2, By further comprising one or more mutations encoding amino acid substitutions at one or more amino acid residues corresponding to amino acid positions 179, 228, 238, 289 or 298, the wild-type aveC allele is inactivated, resulting in mutated nucleotides. The cells of the Streptomyces avermitilis strain ATCC 53692 expressing a polynucleotide molecule comprising the sequence were wild type av A polynucleotide molecule comprising a nucleotide sequence that produces a type 2: 1 ratio of avermectin that differs from that produced by cells of the Streptomyces avermitilis strain ATCC 53692 expressing only the eC allele.
[2" claim-type="Currently amended] The method of claim 1,
The polynucleotide molecule of type 2: 1 avermectin is cyclohexyl B2: cyclohexyl B1 avermectin.
[3" claim-type="Currently amended] The method of claim 2,
A polynucleotide molecule in which the different type 2: 1 ratio of avermectin is a reduced ratio compared to the type 2: 1 ratio produced by the cells of Streptomyces avermitilis strain ATCC 53692 expressing only the wild type aveC allele.
[4" claim-type="Currently amended] The method of claim 3, wherein
A polynucleotide molecule having a ratio of type 2: 1 avermectin up to about 0.8: 1.
[5" claim-type="Currently amended] The method of claim 3, wherein
A polynucleotide molecule having a ratio of type 2: 1 avermectin up to about 0.68: 1.
[6" claim-type="Currently amended] The method of claim 3, wherein
A polynucleotide molecule having a ratio of type 2: 1 avermectin up to about 0.53: 1.
[7" claim-type="Currently amended] The method of claim 3, wherein
A polynucleotide molecule having a ratio of type 2: 1 avermectin up to about 0.42: 1.
[8" claim-type="Currently amended] The method of claim 3, wherein
A polynucleotide molecule having a ratio of type 2: 1 avermectin up to about 0.40: 1.
[9" claim-type="Currently amended] The method of claim 1,
A polynucleotide molecule, wherein the nucleotide sequence further comprises one or more mutations encoding amino acid substitutions at one or both amino acid residues corresponding to amino acid positions 138 and 139 of SEQ ID NO: 2.
[10" claim-type="Currently amended] The method of claim 2,
Amino acid substituents
(a) amino acid residue Q at position 38 replaced by P or an amino acid that is a conservative substituent for P;
(b) E or amino acid residue D at position 48 replaced by an amino acid that is a conservative substituent for E;
(c) amino acid residue A at position 89 replaced by T or an amino acid that is a conservative substituent for T;
(d) amino acid residue F at position 99 replaced by S or an amino acid that is a conservative substituent for S;
(e) amino acid residue G at position 111 replaced by V, or amino acid that is a conservative substituent on V;
(f) P or amino acid residue L at position 136 replaced by amino acid that is a conservative substituent on P;
(g) E or amino acid residue K at position 154 replaced by an amino acid that is a conservative substituent for E;
(h) amino acid residue G at position 179 replaced by S or an amino acid that is a conservative substituent for S;
(i) amino acid residue M at position 228 replaced by T or an amino acid that is a conservative substituent for T;
(j) amino acid residue E at position 238 replaced by D or the amino acid that is a conservative substituent for D;
(k) L, or amino acid residue P at position 289 replaced by amino acid that is a conservative substituent for L; And
(l) a polynucleotide molecule selected from one or more groups consisting of amino acid residue Q at position 298 replaced by an amino acid that is a conservative substituent for H.
[11" claim-type="Currently amended] The method of claim 2,
The combination of amino acid residues is mutated, where the combination
(a) amino acid residues D48 and A89;
(b) amino acid residues S138, A139 and G179;
(c) amino acid residues Q38, L136 and E238;
(d) amino acid residues F99, S138, A139 and G179;
(e) amino acid residues A139 and M228;
(f) amino acid residues G111 and P289; And
(g) A polynucleotide molecule selected from one or more groups consisting of amino acid residues A139, K154 and Q298.
[12" claim-type="Currently amended] The method of claim 11,
Combinations of amino acid substituents include (a) D48E / A89T; (b) S138T / A139T / G179S; (c) Q38P / L136P / E238D; (d) F99S / S138T / A139T / G179S; (e) A139T / M228T; (f) G111V / P289L; And (g) a polynucleotide molecule selected from at least one group consisting of A139T / K154E / Q298H.
[13" claim-type="Currently amended] The method of claim 12,
A mutation in the aveC sequence encoding D48E / A89T results in a base change from T to A at the nucleotide position corresponding to 317 nucleotides of SEQ ID NO: 1, and from G at the nucleotide position corresponding to 438 nucleotides of SEQ ID NO: 1 A polynucleotide molecule comprising a base change to A.
[14" claim-type="Currently amended] The method of claim 13,
A polynucleotide molecule further comprising a base change from C to A at the nucleotide position corresponding to nucleotide 353 of SEQ ID NO: 1, and a base change from T to A at the nucleotide position corresponding to 1155 nucleotide of SEQ ID NO: 1 .
[15" claim-type="Currently amended] The method of claim 12,
A mutation in the aveC sequence encoding S138T / A139T / G179S results in a base change from T to A at the nucleotide position corresponding to nucleotide 585 of SEQ ID NO: 1, G at the nucleotide position corresponding to nucleotide 588 of SEQ ID NO: 1 A nucleotide molecule comprising a nucleotide change from A to A, and a nucleotide change from G to A at the nucleotide position corresponding to 708 nucleotide of SEQ ID NO: 1.
[16" claim-type="Currently amended] The method of claim 15,
A polynucleotide molecule further comprising a base change from G to A at the nucleotide position corresponding to nucleotide 272 of SEQ ID NO: 1.
[17" claim-type="Currently amended] The method of claim 12,
A mutation in the aveC sequence encoding Q38P / L136P / E238D results in a base change from A to C at the nucleotide position corresponding to nucleotide 286 of SEQ ID NO: 1, T at the nucleotide position corresponding to 580 nucleotide of SEQ ID NO: 1 To nucleotide C, and a nucleotide molecule from A to T at the nucleotide position corresponding to 886 nucleotide of SEQ ID NO: 1.
[18" claim-type="Currently amended] The method of claim 17,
Base change from A to G at the nucleotide position corresponding to nucleotide 24 of SEQ ID NO: 1, base change from T to C at the nucleotide position corresponding to 497 nucleotide of SEQ ID NO: 1, and 554 of SEQ ID NO: 1 A polynucleotide molecule further comprising a base change from C to T at the nucleotide position corresponding to the nucleotide.
[19" claim-type="Currently amended] The method of claim 12,
The mutation in the aveC sequence encoding F99S / S138T / A139T / G179S results in three base pair deletions at the nucleotide positions corresponding to nucleotides 173, 174 and 175 of SEQ ID NO: 1, and 469 of SEQ ID NO: 1 Base change from T to C at the nucleotide position corresponding to, nucleotide change from T to A at the nucleotide position corresponding to nucleotide 585 of SEQ ID NO: 1, from G at the nucleotide position corresponding to nucleotide 588 of SEQ ID NO: 1 A polynucleotide molecule comprising a base change from A to a base change from G to A at a nucleotide position corresponding to 708 nucleotide of SEQ ID NO: 1.
[20" claim-type="Currently amended] The method of claim 19,
A polynucleotide molecule further comprising a base change from C to T at the nucleotide position corresponding to nucleotide 833 of SEQ ID NO: 1, and a base change from G to A at the nucleotide position corresponding to nucleotide 1184 of SEQ ID NO: 1 .
[21" claim-type="Currently amended] The method of claim 12,
A mutation in the aveC sequence encoding A139T / M228T results in a base change from G to A at the nucleotide position corresponding to nucleotide 588 of SEQ ID NO: 1 and from T at the nucleotide position corresponding to 856 nucleotide of SEQ ID NO: 1 A polynucleotide molecule comprising a base change to C.
[22" claim-type="Currently amended] The method of claim 12,
A mutation in the aveC sequence encoding G111V / P289L results in a base change from G to T at the nucleotide position corresponding to nucleotide 505 of SEQ ID NO: 1, and from C at the nucleotide position corresponding to nucleotide 1039 in SEQ ID NO: 1 A polynucleotide molecule comprising a base change to T.
[23" claim-type="Currently amended] The method of claim 22,
Base change from T to C at the nucleotide position corresponding to 155 nucleotides of SEQ ID NO: 1, base change from C to T at the nucleotide position corresponding to nucleotides 1202 of SEQ ID NO: 1, and 1210 of SEQ ID NO: 1 A polynucleotide molecule further comprising a base change from T to C at the nucleotide position corresponding to the nucleotide.
[24" claim-type="Currently amended] The method of claim 12,
A mutation in the aveC sequence encoding A139T / K154E / Q298H results in a base change from G to A at the nucleotide position corresponding to nucleotide 588 of SEQ ID NO: 1, A at the nucleotide position corresponding to 633 in SEQ ID NO: 1 A nucleotide molecule comprising a nucleotide change from G to G and a base change from A to T at the nucleotide position corresponding to nucleotide 1067 of SEQ ID NO: 1.
[25" claim-type="Currently amended] The method of claim 24,
A polynucleotide molecule further comprising a base change from G to T at the nucleotide position corresponding to nucleotide 377 of SEQ ID NO: 1.
[26" claim-type="Currently amended] Recombinant vector comprising the polynucleotide molecule of claim 1.
[27" claim-type="Currently amended] A host cell comprising the polynucleotide molecule of claim 1 or the recombinant vector of claim 26.
[28" claim-type="Currently amended] The method of claim 27,
Host cells that are Streptomyces cells.
[29" claim-type="Currently amended] Corresponds to amino acid residues of SEQ ID NO: 2, 38, 48, 89, 99, 111, 136, 138, 139, 154, 179, 228, 238, 289 or 298 AveC alleles of cells of the Streptomyces avermitilis strain causing substitution in the AveC gene product of the different amino acid residues at one or more amino acid positions are mutated so that the aveC allele is thus mutated Streptomyces avermitilis . Streptomyces avermi , wherein the cells of the strain produce a type 2: 1 ratio of avermectin that differs from that produced by cells of the same Streptomyces avermitilis strain expressing only the wild type aveC allele Method for preparing a novel strain of tilis.
[30" claim-type="Currently amended] The method of claim 29,
The type 2: 1 avermectin is cyclohexyl B2: cyclohexyl B1 avermectin.
[31" claim-type="Currently amended] The method of claim 30,
The different type 2: 1 ratio of avermectins is the reduced ratio.
[32" claim-type="Currently amended] The method of claim 31, wherein
The ratio of type 2: 1 avermectin produced by cells of the Streptomyces avermitilis strain mutated aveC allele is no greater than about 0.8: 1.
[33" claim-type="Currently amended] The method of claim 31, wherein
The ratio of type 2: 1 avermectin produced by cells of the Streptomyces avermitilis strain mutated aveC allele is no greater than about 0.68: 1.
[34" claim-type="Currently amended] The method of claim 31, wherein
The ratio of type 2: 1 avermectin produced by cells of the Streptomyces avermitilis strain mutated aveC allele is no greater than about 0.53: 1.
[35" claim-type="Currently amended] The method of claim 31, wherein
The ratio of type 2: 1 avermectin produced by cells of the Streptomyces avermitilis strain mutated aveC allele is about 0.42: 1 or less.
[36" claim-type="Currently amended] The method of claim 31, wherein
The ratio of type 2: 1 avermectin produced by cells of the Streptomyces avermitilis strain mutated aveC allele is about 0.40: 1 or less.
[37" claim-type="Currently amended] The method of claim 31, wherein
Further comprising introducing one or more mutations into the aveC allele encoding an amino acid substitution at one or both amino acid residues corresponding to amino acid positions 138 and 139 of SEQ ID NO: 2.
[38" claim-type="Currently amended] The method of claim 30,
Amino acid substituents
(a) amino acid residue Q at position 38 replaced by P or an amino acid that is a conservative substituent for P;
(b) E or amino acid residue D at position 48 replaced by an amino acid that is a conservative substituent for E;
(c) amino acid residue A at position 89 replaced by T or an amino acid that is a conservative substituent for T;
(d) amino acid residue F at position 99 replaced by S or an amino acid that is a conservative substituent for S;
(e) amino acid residue G at position 111 replaced by V, or amino acid that is a conservative substituent on V;
(f) P or amino acid residue L at position 136 replaced by amino acid that is a conservative substituent on P;
(g) E or amino acid residue K at position 154 replaced by an amino acid that is a conservative substituent for E;
(h) amino acid residue G at position 179 replaced by S or an amino acid that is a conservative substituent for S;
(i) amino acid residue M at position 228 replaced by T or an amino acid that is a conservative substituent for T;
(j) amino acid residue E at position 238 replaced by D or the amino acid that is a conservative substituent for D;
(k) L, or amino acid residue P at position 289 replaced by amino acid that is a conservative substituent for L; And
(l) H, or a method selected from the group consisting of amino acid residues Q at position 298 replaced by amino acids that are conservative substituents on H.
[39" claim-type="Currently amended] The method of claim 30,
the aveC allele is mutated to encode an amino acid substituent at a combination of amino acid positions, wherein the combination
(a) amino acid residues D48 and A89;
(b) amino acid residues S138, A139 and G179;
(c) amino acid residues Q38, L136 and E238;
(d) amino acid residues F99, S138, A139 and G179;
(e) amino acid residues A139 and M228;
(f) amino acid residues G111 and P289; And
(g) a method selected from one or more groups consisting of amino acid residues A139, K154 and Q298.
[40" claim-type="Currently amended] The method of claim 39,
Combinations of amino acid substituents include (a) D48E / A89T; (b) S138T / A139T / G179S; (c) Q38P / L136P / E238D; (d) F99S / S138T / A139T / G179S; (e) A139T / M228T; (f) G111V / P289L; And (g) A139T / K154E / Q298H.
[41" claim-type="Currently amended] The method of claim 40,
The mutation in the aveC allele encoding D48E / A89T corresponds to a base change from T to A at the nucleotide position of the aveC allele corresponding to 317 nucleotides of SEQ ID NO: 1, and corresponding to 438 nucleotides of SEQ ID NO: 1 a base change from G to A at the nucleotide position of the aveC allele.
[42" claim-type="Currently amended] 42. The method of claim 41 wherein
Base change from C to A at the nucleotide position of the aveC allele corresponding to nucleotide 353 of SEQ ID NO: 1, and base change from T to A at the nucleotide position of the aveC allele corresponding to 1155 nucleotide of SEQ ID NO: 1 The method further comprises introducing.
[43" claim-type="Currently amended] The method of claim 40,
A mutation in the aveC allele encoding S138T / A139T / G179S corresponds to a nucleotide change from T to A at the nucleotide position of the aveC allele corresponding to nucleotide 585 of SEQ ID NO: 1, corresponding to nucleotide 588 of SEQ ID NO: 1 A base change from G to A at the nucleotide position of the aveC allele, and a base change from G to A at the nucleotide position of the aveC allele corresponding to 708 nucleotide of SEQ ID NO: 1.
[44" claim-type="Currently amended] The method of claim 43,
And introducing a base change from G to A at the nucleotide position of the aveC allele corresponding to 272 nucleotide of SEQ ID NO: 1.
[45" claim-type="Currently amended] The method of claim 40,
A mutation in the aveC allele encoding Q38P / L136P / E238D corresponds to a nucleotide change from A to C at the nucleotide position of the aveC allele corresponding to nucleotide 286 of SEQ ID NO: 1, corresponding to 580 nucleotide of SEQ ID NO: 1 A base change from T to C at the nucleotide position of the aveC allele, and a base change from A to T at the nucleotide position of the aveC allele corresponding to 886 number nucleotide of SEQ ID NO: 1.
[46" claim-type="Currently amended] The method of claim 45,
Base change from A to G at the nucleotide position of the aveC allele corresponding to nucleotide 24 of SEQ ID NO: 1, base change from T to C at the nucleotide position of the aveC allele corresponding to 497 nucleotide of SEQ ID NO: 1, And introducing a base change from C to T at the nucleotide position of the aveC allele corresponding to 554 nucleotide of SEQ ID NO: 1.
[47" claim-type="Currently amended] The method of claim 40,
The mutation in the aveC allele encoding F99S / S138T / A139T / G179S results in three base pair deletions at the nucleotide position of the aveC allele corresponding to nucleotides 173, 174 and 175 of SEQ ID NO: 1, SEQ ID NO: Base change from T to C at the nucleotide position of the aveC allele corresponding to 469 nucleotides of 1, base change from T to A at the nucleotide position of the aveC allele corresponding to nucleotide 585 of SEQ ID NO: 1, SEQ ID NO: A base change from G to A at the nucleotide position of the aveC allele corresponding to nucleotide 588 of 1, and a base change from G to A at the nucleotide position of the aveC allele corresponding to 708 nucleotide of SEQ ID NO: 1 Way.
[48" claim-type="Currently amended] The method of claim 47,
Base change from C to T at the nucleotide position of the aveC allele corresponding to 833 nucleotide of SEQ ID NO: 1, and base change from G to A at the nucleotide position of the aveC allele corresponding to 1184 nucleotide of SEQ ID NO: 1 The method further comprises introducing.
[49" claim-type="Currently amended] The method of claim 40,
The mutation in the aveC allele encoding A139T / M228T corresponds to a base change from G to A at the nucleotide position of the aveC allele corresponding to nucleotide 588 of SEQ ID NO: 1, and corresponding to 856 nucleotide of SEQ ID NO: 1 and a base change from T to C at the nucleotide position of the aveC allele.
[50" claim-type="Currently amended] The method of claim 40,
The mutation in the aveC allele encoding G111V / P289L corresponds to a base change from G to T at the nucleotide position of the aveC allele corresponding to nucleotide 505 of SEQ ID NO: 1, and corresponding to nucleotide 1039 of SEQ ID NO: 1 a base change from C to T at the nucleotide position of the aveC allele.
[51" claim-type="Currently amended] 51. The method of claim 50 wherein
Base change from T to C at the nucleotide position of the aveC allele corresponding to 155 nucleotide of SEQ ID NO: 1, base change from C to T at the nucleotide position of the aveC allele corresponding to nucleotide 1202 of SEQ ID NO: 1, And introducing a base change from T to C at the nucleotide position of the aveC allele corresponding to nucleotide 1210 of SEQ ID NO: 1.
[52" claim-type="Currently amended] The method of claim 40,
A mutation in the aveC allele encoding A139T / K154E / Q298H corresponds to a nucleotide change from G to A at the nucleotide position of the aveC allele corresponding to nucleotide 588 of SEQ ID NO: 1, nucleotide 633 of SEQ ID NO: 1 A base change from A to G at the nucleotide position of the aveC allele, and a base change from A to T at the nucleotide position of the aveC allele corresponding to 1067 nucleotide of SEQ ID NO: 1.
[53" claim-type="Currently amended] The method of claim 52, wherein
Further comprising introducing a base change from G to T at the nucleotide position of the aveC allele corresponding to nucleotide 377 of SEQ ID NO: 1.
[54" claim-type="Currently amended] At one or more amino acid positions corresponding to amino acid residues 38, 48, 89, 99, 111, 136, 154, 179, 228, 238, 289 or 298 of SEQ ID NO: 2 Of avermectins that have a mutated aveC allele that encodes an AveC gene product with substitutions and that are different from the rate produced by the cells of the same Streptomyces avermitilis strain except expressing only the wild type aveC allele Streptomyces avermitilis cells producing a type 2: 1 ratio.
[55" claim-type="Currently amended] The method of claim 54, wherein
Streptomyces avermitilis cells, wherein the type 2: 1 avermectin is cyclohexyl B2: cyclohexyl B1 avermectin.
[56" claim-type="Currently amended] The method of claim 55,
Streptomyces avermitilis cells in which the different type 2: 1 ratio of avermectin is reduced.
[57" claim-type="Currently amended] The method of claim 56, wherein
Streptomyces avermitilis cells producing a ratio of type 2: 1 avermectin up to about 0.8: 1.
[58" claim-type="Currently amended] The method of claim 56, wherein
Streptomyces avermitilis cells producing a ratio of type 2: 1 avermectin up to about 0.68: 1.
[59" claim-type="Currently amended] The method of claim 56, wherein
Streptomyces avermitilis cells producing a ratio of type 2: 1 avermectin up to about 0.53: 1.
[60" claim-type="Currently amended] The method of claim 56, wherein
Streptomyces avermitilis cells producing a ratio of type 2: 1 avermectin up to about 0.42: 1.
[61" claim-type="Currently amended] The method of claim 56, wherein
Streptomyces avermitilis cells producing a ratio of type 2: 1 avermectin up to about 0.40: 1.
[62" claim-type="Currently amended] The method of claim 55,
Streptomyces avermitilis cells, wherein the aveC allele further encodes amino acid substitutions at one or both amino acid residues corresponding to amino acid positions 138 and 139 of SEQ ID NO: 2.
[63" claim-type="Currently amended] The method of claim 55,
Amino acid substituents
(a) amino acid residue Q at position 38 replaced by P or an amino acid that is a conservative substituent for P;
(b) E or amino acid residue D at position 48 replaced by an amino acid that is a conservative substituent for E;
(c) amino acid residue A at position 89 replaced by T or an amino acid that is a conservative substituent for T;
(d) amino acid residue F at position 99 replaced by S or an amino acid that is a conservative substituent for S;
(e) amino acid residue G at position 111 replaced by V or an amino acid that is a conservative substituent for V;
(f) P, or amino acid residue L at position 136 replaced by amino acid that is a conservative substituent for P;
(g) E or amino acid residue K at position 154 replaced by an amino acid that is a conservative substituent for E;
(h) amino acid residue G at position 179 replaced by S or an amino acid that is a conservative substituent for S;
(i) amino acid residue M at position 228 replaced by T or an amino acid that is a conservative substituent for T;
(j) amino acid residue E at position 238 replaced by D or the amino acid that is a conservative substituent for D;
(k) L, or amino acid residue P at position 289 replaced by amino acid that is a conservative substituent for L; And
(l) Streptomyces avermitilis cells selected from one or more groups consisting of amino acid residues Q at position 298 replaced by H or an amino acid that is a conservative substituent for H.
[64" claim-type="Currently amended] The method of claim 55,
the aveC allele is mutated to encode an amino acid substituent at a combination of amino acid positions, wherein the combination
(a) amino acid residues D48 and A89;
(b) amino acid residues S138, A139 and G179;
(c) amino acid residues Q38, L136 and E238;
(d) amino acid residues F99, S138, A139 and G179;
(e) amino acid residues A139 and M228;
(f) amino acid residues G111 and P289; And
(g) Streptomyces avermitilis cells selected from one or more groups consisting of amino acid residues A139, K154 and Q298.
[65" claim-type="Currently amended] The method of claim 64, wherein
Combinations of amino acid substituents include (a) D48E / A89T; (b) S138T / A139T / G179S; (c) Q38P / L136P / E238D; (d) F99S / S138T / A139T / G179S; (e) A139T / M228T; (f) G111V / P289L; And (g) Streptomyces avermitilis cells selected from one or more groups consisting of A139T / K154E / Q298H.
[66" claim-type="Currently amended] 66. The method of claim 65,
The mutation in the aveC allele encoding D48E / A89T corresponds to a base change from T to A at the nucleotide position of the aveC allele corresponding to 317 nucleotides of SEQ ID NO: 1, and corresponding to 438 nucleotides of SEQ ID NO: 1 Streptomyces avermitilis cells comprising a base change from G to A at the nucleotide position of the aveC allele.
[67" claim-type="Currently amended] The method of claim 66, wherein
Base change from C to A at the nucleotide position of the aveC allele corresponding to nucleotide 353 of SEQ ID NO: 1, and base change from T to A at the nucleotide position of the aveC allele corresponding to 1155 nucleotide of SEQ ID NO: 1 Streptomyces avermitilis cells further comprising.
[68" claim-type="Currently amended] 66. The method of claim 65,
A mutation in the aveC allele encoding S138T / A139T / G179S corresponds to a nucleotide change from T to A at the nucleotide position of the aveC allele corresponding to nucleotide 585 of SEQ ID NO: 1, corresponding to nucleotide 588 of SEQ ID NO: 1 Streptomyces avermitilis comprising a base change from G to A at the nucleotide position of the aveC allele, and a base change from G to A at the nucleotide position of the aveC allele corresponding to 708 nucleotide of SEQ ID NO: 1 cell.
[69" claim-type="Currently amended] The method of claim 68, wherein
A mutation in the aveC allele encoding S138T / A139T / G179S further comprises a base change from G to A at the nucleotide position of the aveC allele corresponding to 272 nucleotides of SEQ ID NO: 1 S cells.
[70" claim-type="Currently amended] 66. The method of claim 65,
A mutation in the aveC allele encoding Q38P / L136P / E238D corresponds to a nucleotide change from A to C at the nucleotide position of the aveC allele corresponding to nucleotide 286 of SEQ ID NO: 1, corresponding to 580 nucleotide of SEQ ID NO: 1 Streptomyces avermitilis comprising a base change from T to C at the nucleotide position of the aveC allele and a base change from A to T at the nucleotide position of the aveC allele corresponding to 886 number nucleotide of SEQ ID NO: 1 cell.
[71" claim-type="Currently amended] The method of claim 70,
A mutation in the aveC allele encoding Q38P / L136P / E238D corresponds to a nucleotide change from A to G at the nucleotide position of the aveC allele corresponding to nucleotide 24 of SEQ ID NO: 1, corresponding to nucleotide 497 of SEQ ID NO: 1 Streptomyces avermi further comprising a base change from T to C at the nucleotide position of the aveC allele and a base change from C to T at the nucleotide position of the aveC allele corresponding to 554 nucleotide of SEQ ID NO: 1 Tilis cells.
[72" claim-type="Currently amended] 66. The method of claim 65,
The mutation in the aveC allele encoding F99S / S138T / A139T / G179S results in three base pair deletions at the nucleotide position of the aveC allele corresponding to nucleotides 173, 174 and 175 of SEQ ID NO: 1, SEQ ID NO: Base change from T to C at the nucleotide position of the aveC allele corresponding to 469 nucleotides of 1, base change from T to A at the nucleotide position of the aveC allele corresponding to nucleotide 585 of SEQ ID NO: 1, SEQ ID NO: A base change from G to A at the nucleotide position of the aveC allele corresponding to nucleotide 588 of 1, and a base change from G to A at the nucleotide position of the aveC allele corresponding to 708 nucleotide of SEQ ID NO: 1 Streptomyces avermitilis cells.
[73" claim-type="Currently amended] The method of claim 72,
The mutation in the aveC allele encoding F99S / S138T / A139T / G179S results in a base change from C to T at the nucleotide position of the aveC allele corresponding to nucleotide 833 of SEQ ID NO: 1, and 1184 of SEQ ID NO: 1 A Streptomyces avermitilis cell, further comprising a base change from G to A at the nucleotide position of the aveC allele corresponding to the nucleotide.
[74" claim-type="Currently amended] 66. The method of claim 65,
The mutation in the aveC allele encoding A139T / M228T corresponds to a base change from G to A at the nucleotide position of the aveC allele corresponding to nucleotide 588 of SEQ ID NO: 1, and corresponding to 856 nucleotide of SEQ ID NO: 1 Streptomyces avermitilis cells comprising a base change from T to C at the nucleotide position of the aveC allele.
[75" claim-type="Currently amended] 66. The method of claim 65,
The mutation in the aveC allele encoding G111V / P289L corresponds to a base change from G to T at the nucleotide position of the aveC allele corresponding to nucleotide 505 of SEQ ID NO: 1, and corresponding to nucleotide 1039 of SEQ ID NO: 1 Streptomyces avermitilis cells comprising a base change from C to T at the nucleotide position of the aveC allele.
[76" claim-type="Currently amended] 76. The method of claim 75 wherein
A mutation in the aveC allele encoding G111V / P289L results in a nucleotide change from T to C at the nucleotide position of the aveC allele corresponding to 155 nucleotides of SEQ ID NO: 1, aveC corresponding to nucleotide 1202 of SEQ ID NO: 1 Streptomyces avermitilis further comprising a base change from C to T at the nucleotide position of the allele and a base change from T to C at the nucleotide position of the aveC allele corresponding to nucleotide 1210 of SEQ ID NO: 1 cell.
[77" claim-type="Currently amended] 66. The method of claim 65,
A mutation in the aveC allele encoding A139T / K154E / Q298H corresponds to a nucleotide change from G to A at the nucleotide position of the aveC allele corresponding to nucleotide 588 of SEQ ID NO: 1, nucleotide 633 of SEQ ID NO: 1 Streptomyces avermitilis comprising a base change from A to G at the nucleotide position of the aveC allele and a base change from A to T at the nucleotide position of the aveC allele corresponding to nucleotide 1067 of SEQ ID NO: 1 cell.
[78" claim-type="Currently amended] 78. The method of claim 77 wherein
The mutation in the aveC allele encoding A139T / K154E / Q298H further comprises a base change from G to T at the nucleotide position of the aveC allele corresponding to nucleotide 377 of SEQ ID NO: 1 S cells.
[79" claim-type="Currently amended] Culturing the cells of claim 55 in a medium under conditions permitting or inducing production of avermectin; And recovering avermectin from the culture solution.
[80" claim-type="Currently amended] Of cyclohexyl B2: cyclohexyl B1 avermectin produced by cells of Streptomyces avermitilis comprising cyclohexyl B2: cyclohexyl B1 avermectin in a cell culture medium at a ratio of about 0.68: 1 or less. Composition.
[81" claim-type="Currently amended] Type 2 of cyclohexyl B2: cyclohexyl B1 avermectin produced by the cell compared to cells of the same strain of Streptomyces avermitilis that do not express the mutated aveC allele but express only the wild type aveC allele. Expresses a mutated aveC allele encoding a gene product that reduces the rate by 1,
Cyclohexyl B2: cyclohexyl B1 avermectin produced by cells of a Streptomyces avermitilis strain comprising cyclohexyl B2: cyclohexyl B1 avermectin in a ratio of about 0.68: 1 or less in the cell culture medium Composition.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Faust et al.2000|Two new tailoring enzymes, a glycosyltransferase and an oxygenase, involved in biosynthesis of the angucycline antibiotic urdamycin A in Streptomyces fradiae Tü2717This paper is dedicated to Professor Heinz Floss, a pioneer in the field of antibiotics, on the occasion of his 65th birthday. The GenBank accession numbers for the sequences reported in this paper are AF164960 and AF164961.

---

US6200813B1|2001-03-13|Polyketide derivatives and recombinant methods for making same

---

US6670168B1|2003-12-30|Recombinant Streptomyces hygroscopicus host cells that produce 17-desmethylrapamycin

---

US8624009B2|2014-01-07|Spinosyn-producing polyketide synthases

---

US6521406B1|2003-02-18|SpnG, a gene for spinosyn insecticide biosynthesis

---

US5141926A|1992-08-25|Erythromycin derivatives

---

ES2287552T3|2007-12-16|Derivatives of borrelidine and its use in medicine.

---

Huang et al.2009|Recent advances in the biochemistry of spinosyns

---

US6251636B1|2001-06-26|Recombinant oleandolide polyketide synthase

---

Bender et al.1993|Characterization of the genes controlling the biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid

---

Jin et al.2009|Enhanced production of spinosad in Saccharopolyspora spinosa by genome shuffling

---

US6399324B1|2002-06-04|Genes encoding branched-chain alpha-ketoacid dehydrogenase complex from Streptomyces avermitilis

---

KR20010085877A|2001-09-07|Polyketide synthase enzymes and recombinant dna constructs therefor

---

Qiu et al.2011|Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis

---

JP2008022865A|2008-02-07|Production of polyketides in bacteria and yeast

---

KR20010071684A|2001-07-31|Polyketides and their synthesis

---

Kitani et al.2009|Characterization of a regulatory gene, aveR, for the biosynthesis of avermectin in Streptomyces avermitilis

---

Stutzman-Engwall et al.2005|Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis

---

CA2259463A1|1998-01-15|Polyketides and their synthesis

---

HU217208B|1999-12-28|Method for producing phosphinothricin resistence gene

---

US20030104585A1|2003-06-05|Polyketides and their synthesis and use

---

JP2674998B2|1997-11-12|Improvement of antibiotic-producing microorganisms

---

EP0284176B1|1993-08-25|Process for production of avermectins and cultures therefor

---

Liu et al.2006|Identification of NanE as the thioesterase for polyether chain release in nanchangmycin biosynthesis

---

US8148102B2|2012-04-03|Sequences for FK228 biosynthesis and methods of synthesizing FK228 and FK228 analogs

同族专利:

---

公开号 | 公开日

---

UY26286A1|2001-03-16|

---

NZ516517A|2003-10-31|

---

BG106479A|2002-10-31|

---

AP200202418A0|2002-03-31|

---

CN1373809A|2002-10-09|

---

HK1046710B|2005-05-13|

---

ES2272299T3|2007-05-01|

---

KR100489856B1|2005-05-17|

---

CN1186446C|2005-01-26|

---

EA200200056A1|2002-06-27|

---

US6632673B1|2003-10-14|

---

NO20020664D0|2002-02-11|

---

HRP20020130A2|2003-06-30|

---

HU0202487A3|2004-07-28|

---

CO5290295A1|2003-06-27|

---

IL147563A|2009-06-15|

---

HK1046710A1|2005-05-13|

---

DE60031126D1|2006-11-16|

---

PA8499501A1|2001-12-14|

---

OA11998A|2006-04-18|

---

HU230600B1|2017-02-28|

---

DE60031126T2|2007-02-08|

---

ZA200201072B|2003-04-30|

---

MXPA02001513A|2002-07-02|

---

JP2003507017A|2003-02-25|

---

PT1200606E|2006-12-29|

---

SK288086B6|2013-06-03|

---

JP3884651B2|2007-02-21|

---

US20030232415A1|2003-12-18|

---

IS6250A|2002-01-25|

---

EP1200606A1|2002-05-02|

---

BG65902B1|2010-04-30|

---

US7858340B2|2010-12-28|

---

AU778837B2|2004-12-23|

---

BR0013119A|2002-04-23|

---

PL203818B1|2009-11-30|

---

WO2001012821A1|2001-02-22|

---

NO20020664L|2002-02-11|

---

SK1392002A3|2002-12-03|

---

AT341632T|2006-10-15|

---

EP1200606B1|2006-10-04|

---

PL353874A1|2003-12-01|

---

PE20010690A1|2001-07-06|

---

GT200000136A|2002-02-01|

---

AU5838600A|2001-03-13|

---

MA26813A1|2004-12-20|

---

IL147563D0|2002-08-14|

---

CZ304402B6|2014-04-16|

---

CZ2002296A3|2002-08-14|

---

HU0202487A2|2002-10-28|

---

CA2380872A1|2001-02-22|

---

BR0013119B1|2014-02-25|

---

CA2380872C|2007-11-27|

---

DK1200606T3|2007-01-08|

---

TNSN00171A1|2005-11-10|

---

AR022646A1|2002-09-04|

---

US20040009560A1|2004-01-15|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
1999-08-12|Priority to US14864599P

---

1999-08-12|Priority to US60/148,645

---

2000-07-24|Application filed by 실버스타인 아써 에이., 화이자 프로덕츠 인코포레이티드

---

2002-04-18|Publication of KR20020029379A

---

2005-05-17|Application granted

---

2005-05-17|Publication of KR100489856B1

优先权:

---

申请号 | 申请日 | 专利标题

---

US14864599P| true| 1999-08-12|1999-08-12|

---

US60/148,645|1999-08-12|