Process for the Biological Production of 1,3-Propanediol

专利摘要:
The present invention provides an improved method for biologically producing 1,3-propanediol from a carbon source fermentable in a single microorganism. In one aspect of the invention, improved methods for converting glucose to 1,3-propanediol include the Klebsiella pneumoniae dha regulatory genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, And E. coli transformed with orfZ (all of these genes are arranged in the same genetic tissue as found in wild type Klebsiella pneumoniae). In another aspect of the invention, a method of using recombinant E. coli containing genes encoding G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factor to produce 1,3-propanediol from glucose comprises It is an improvement over the same method using recombinant E. coli, which contains genes encoding 1,3-propanediol oxidoreductase (dhaT) as well as G3P phosphatase, dehydratase, dehydratase reactivation factor. The dramatic improvement of this method depends on the presence or absence of endogenous genes encoding enough nonspecific catalytic activity in E. coli to convert 3-hydroxypropionaldehyde to 1,3-propanediol.
公开号:KR20020059364A
申请号:KR1020027002027
申请日:2000-08-18
公开日:2002-07-12
发明作者:마크 엠프테이지；샤론 헤이니；리사 라펜드；제프 푸시；그레그 화이티드
申请人:메리 이. 보울러；이 아이 듀폰 디 네모아 앤드 캄파니；마가렛 에이.혼；제넨코 인터내셔날 인코포레이티드；
IPC主号:

专利说明:

Process for the Biological Production of 1,3-Propanediol
[2] 1,3-propanediol is a monomer of potential utility in polyester fiber production and in the preparation of polyurethanes and cyclic compounds.
[3] Various chemical routes to produce 1,3-propanediol are known. For example, ethylene oxide is reduced after catalytic solution phase hydration of acrolein on the catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and acid, or on a catalyst having a Group VIII element of the periodic table from a compound such as glycerol and It can be converted to 1,3-propanediol by reaction in the presence of hydrogen. Although 1,3-propanediol can be produced in this way, this method is expensive and generates waste streams containing environmental pollutants.
[4] The fact that fermentation of glycerol to produce 1,3-propanediol has been known for over a century. 1,3-propane been found that bacterial strains which can produce a diol, for example, as a sheet bakteo (Citrobacter), Clostridium (Clostridium), Enterobacter bakteo (Enterobacter), one Rio bakteo (Ilyobacter), keulrep when Ella ( Klebsiella ), Lactobacillus , and Pelobacter family. In each case, glycerol is converted to 1,3-propanediol via a two stage enzymatic catalysis. In the first step, dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) and water (Scheme 1). In a second step, 3-HPA is reduced to 1,3-propanediol by NAD ⁺ -linked oxidoreductase (Scheme 2). 1,3-propanediol is no longer metabolized and consequently accumulates in the medium. In the overall reaction, the cofactor β-nicotinamide adenine dinucleotide (NADH) is consumed in one reduced equivalent in reduced form and oxidized to nicotinamide adenine dinucleotide (NAD ⁺ ).
[5] Glycerol → 3-HPA + H ₂ 0
[6] 3-HPA + NADH + H ⁺ → 1,3-propanediol + NAD ⁺
[7] Klebsiella pneumoniae (Klebsiella pneumonia), Citrobacter Prodydy (Citrobacter freundii), And Clostridium pasteerianum (Clostridium pasteurianum), The gene encoding the three structural subunits of glycerol dehydratase (dhaB1-3 or dhaB, C and E) is adjacent to the gene encoding specific 1,3-propanediol oxidoreductase (dhaT) (See Fig. 1). The genetic organization between these microorganisms is somewhat different, but these genes are orfX and orfZ (the genes encoding dehydratase reactivation factor for glycerol dehydratase), and orfY and orfW (of these genes) Functions are clustered into a group that also includes unknown). Specific 1,3-propanediol oxidoreductases (products of dhaT) of these microorganisms are known to belong to the type III alcohol dehydrogenase family; Iron-binding motifs are conserved in each, and NAD of 1,3-propanediol and 3-HPA⁺It is primarily involved in / NADH-binding interconversion. NAD of 1,3-propanediol and 3-HPA⁺/ NADH-binding interconversion has no particular association with dehydratase enzyme It may also be catalyzed by alcohol dehydrogenase (eg horse liver and confectionary yeast alcohol dehydrogenase (E.C.1.1.1.1)), but its kinetic parameters are less effective. Glycerol dehydratase (E.C.4.2.1.30) and diol [1,2-propanediol] dehydratase (E.C.4.2.1.28) are related enzymes but are other enzymes encoded by other genes. Klebsiella oxytoca (Klebsiella oxytoca) And Salmonella typhimurium (Salmonella typhimuriumDiol dehydratase gene is similar to the glycerol dehydratase gene and clustered in a group containing genes similar to orfX and orfZ (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999)). And Toraya and Mori, J. Biol. Chem. 274, 3372 (1999), GenBank AF026270).
[8] 1,3-propanediol production from glycerol typically occurs in the absence of other exogenous reduction equivalent receptors under anaerobic conditions using glycerol as the only carbon source. For example, in Citrobacter, Clostridium, and Klebsiella strains, similar pathways for glycerol operate under these conditions, first of all by glycerol by NAD ⁺ (or NADP ⁺ ) -linked glycerol dehydrogenase. Oxidized to dihydroxyacetone (DHA) (Scheme 3). The DHA is then phosphorylated by DHA kinase to dihydroxyacetone phosphate (DHAP) (Scheme 4), which can be used for the aid and biosynthesis of ATP production through glycolysis and the like. In contrast to the 1,3-propanediol pathway, this pathway can provide carbon and energy to the cell, producing NADH rather than consuming NADH.
[9] Glycerol + NAD ⁺ → DHA + NADH + H ⁺
[10] DHA + ATP → DHAP + ADP
[11] In Klebsiella pneumoniae and Citrobacter proindei, genes encoding functionally linked active glycerol dehydratase (dhaB), genes encoding 1,3-propanediol oxidoreductase (dhaT), glycerol Genes that encode dehydrogenase (dhaD), and genes that encode dihydroxyacetone kinase (dhaK) are encompassed by dha regulon. The dharegulators of Klebsiella pneumoniae and Citrobacter proindide also include a gene encoding the transcriptional activator protein (dhaR). When dha regulators of Citrobacter and Klebsiella were expressed in E. coli, it was shown to convert glycerol to 1,3-propanediol.
[12] Since the chemical method is energy intensive and the biological method has a relatively low titer from glycerol, an expensive starting material, both the chemical and biological methods described above are suitable for industrial scale production of 1,3-propanediol. Not. These drawbacks can be overcome by using methods that require low energy input, low cost starting materials such as carbohydrates or sugars, or by increasing the metabolic efficiency of glycerol processing. Either way, the manipulation of the genetic machinery responsible for converting sugars to glycerol and glycerol to 1,3-propanediol will be required.
[13] Biological methods of preparing glycerol are known. The overwhelming majority of glycerol producers are yeast, but some bacteria and other fungi and algae are also known. Both bacteria and yeast convert glucose or other carbohydrates through the fructose-1, 6-bisphosphate pathway, or the Emb (Membden Meyerhof Parnas) pathway during glycolysis to produce glycerol, but some algae are carbon dioxide or bicarbonate dissolved in chloroplasts. Is converted to a 3-carbon intermediate in the Calvin cycle. In a series of steps, the 3-carbon intermediate, phosphoglyceric acid, is converted to glyceraldehyde 3-phosphate, which can be easily converted to its keto isomer dihydroxyacetone phosphate and ultimately to glycerol.
[14] Specifically, the bacterium Bacillus licheniformis and Lactobacillus lycopersica synthesize glycerol, Dunaliella spp. And asteromonas grassil, which are salt tolerant algae Lys ( Asteromonas gracilis ) has been shown to produce glycerol in order to be protected from external high salt concentrations. Similarly, various osmotic yeasts synthesize glycerol as a protective means. Most of the Saccharomyces strains produce some glycerol during alcoholic fermentation, which may be physiologically increased under osmotic stress. In the early 20th century, commercial production of glycerol was achieved using Saccharomyces cultures with the addition of "steering reagents" such as sulfites or alkalis. Since the steering agent blocks or inhibits the conversion of acetaldehyde to ethanol by forming an inert complex, excess reduction equivalents (NADH) become available for glycerol production or “steer” in the direction of reducing DHAP. This method is limited because of the partial inhibition of yeast growth due to sulfite. This limitation can be partially overcome by using alkalis that produce excess NADH equivalents by different mechanisms. In this practice, the alkali initiates Cannizarro disproportionation to produce ethanol and acetic acid from two equivalents of acetaldehyde.
[15] The genes encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) of Saccharomyces diastaticus were cloned and sequenced [Wang et al., J. Bact. 176, 7091-7095 (1994). The DARI gene was cloned into a shuttle vector and used to transform E. coli and express it to produce an active enzyme. Wang et al., Found that DAR1 is regulated by the intracellular osmotic environment, but suggests how this gene can be used to improve the production of 1,3-propanediol in recombinant microorganisms. I couldn't.
[16] Other glycerol-3-phosphate dehydrogenase enzymes were isolated: for example, sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae was cloned and sequenced (Larason et al., Mol. Microbiol. 10, 1101 (1993), and Albertyn et al., Mol. Cell. Biol. 14, 4135 (1994)], glycerol-3-phosphate of Saccharomyces cerevisiae. The cloning of GPD 1 encoding dehydrogenase is taught. As in Wang et al., Supra, both Albertyn et al. And Lason et al., Supra, indicate that this gene is regulated by osmosensitivity. It has been found, but it has not been suggested how the gene could be used to produce 1,3-propanediol in recombinant microorganisms.
[17] As in the case of G3PDH, glycerol-3-phosphatase was isolated from Saccharomyces cerevisiae, which protein was found to be encoded by the GPP1 and GPP2 genes [Norbeck et al., J Biol. Chem. 271, 13875 (1996). Like the gene encoding G3PDH, GPP2 appears to be osmosensitive.
[18] While it is desirable to convert other fermentable carbon sources other than glycerol or dihydroxyacetone to 1,3-propanediol by single microorganisms, it has been documented that there are significant difficulties to overcome in this attempt. See, eg, Gottschalk et al. (EP 373 230) describes most of the strains useful for the production of 1,3-propanediol, for example Citrobacter proindy, Clostridium autobutylicum. (Clostridium autobutylicum), Clostridium Butylcum (Clostridium butylicum), And the proliferation of Klebsiella pneumoniae and the like is hampered by the presence of hydrogen donors such as fructose or glucose. Lactobacillus brevis (Lactobacillus brevis) And Lactobacillus bukneri (Lactobacillus buchneriStrains produce 1,3-propanediol upon co-fermentation of glycerol and fructose or glucose but do not proliferate when glycerol is provided as the only carbon source, and their resting cells are glucose or fructose It can metabolize toss but does not produce 1,3-propanediol (Veiga DA Cunha et al., J. Bacteriol., 174, 1013 (1992)). Similarly, Iliobacter polytropus (Ilyobacter polytropusStrains produce 1,3-propanediol when provided with glycerol and acetate, but fail to produce 1,3-propanediol from carbon substrates other than glycerol such as fructose and glucose [Steib et al. Arch. Microbiol. 140, 139 (1984). Finally, Ton et al., Appl. Biochem. Biotech. 34, 149 (1992) teaches that recombinant E. coli transformed with dha regulators encoding glycerol dehydratase does not produce 1,3-propanediol from glucose or xylose in the absence of exogenous glycerol.
[19] In an attempt to improve the yield of 1,3-propanediol from glycerol, methods have been reported to include auxiliary substrates, typically fermentable sugars, which may provide a reducing equivalent. Yield improvement method using simultaneous fermentation of glycerol and glucose by resting cells of Citrobacter Prodydy and Klebsiella pneumoniae DSM 4270 has been reported (Gottschalk et al., Supra) and [Tran-Dinh et al. ., DE 3734 764]), simultaneous fermentation of glycerol and glucose by proliferating cells of Klebsiella pneumoniae ATCC 25955 reported no production of 1,3-propanediol [IT. Tong, Ph.D. Thesis, University of Wisconsin-Madison (1992). It has been reported that simultaneous fermentation of glycerol and glucose or fructose by recombinant E. coli increased the yield, but no 1,3-propanediol was produced without glycerol [Tong et al., Supra]. In this system, the microorganism alone uses carbohydrates as a source for NADH production, while using it to provide energy and carbon for cell maintenance or proliferation. This fact means that sugars do not enter the carbon stream producing 1,3-propanediol.
[20] Recently, however, glycerol was produced by a single microorganism expressing the dehydratase enzyme in the literature (US Pat. No. 5,686,276), WO 9821339, WO 9928480, and WO 9821341 (US Pat. No. 6013494). Or it is described that carbon substrates other than dihydroxyacetone are converted to 1,3-propanediol. Certain deficiencies in the biological process in which 1,3-propanediol is produced from glycerol or glucose have low titers of product through fermentation, requiring an energy intensive separation process to obtain 1,3-propanediol from aqueous fermentation broth. . The final titer when producing 1,3-propanediol from glycerol via Fed-batch or batch fermentation was 65 g / L with Clostridium butyricum and [Saint- Amans et al., Biotechnology Letters 16, 831 (1994)] and 71 g / L due to the Clostridium butyricum mutant [Abbad-Andaloussi et al., Appl. Environ. Microbiol. 61, 4413 (1995)], and 61 g / L for Klebsiella pneumoniae, as described by Homann et al., Appl. Bicrobiol. Biotechnol. 33, 121 (1990)], and 35 g / L with Citrobacter Prodydye (Homann et al., Supra). The titer when fermenting glucose to produce 1,3-propanediol exceeds the titer obtained from glycerol fermentation has not yet been disclosed.
[21] Biological production methods for the production of high titers of 1,3-propanediol from low cost carbon substrates such as glucose or other sugars using a single microorganism are still a challenge. Biological production of 1,3-propanediol is a two-step sequential reaction that requires glycerol as a substrate, where the dehydratase enzyme (typically coenzyme B ₁₂ -dependent dehydratase) is an intermediate of glycerol. After conversion to hydroxypropionaldehyde, it is reduced to 1,3-propanediol by NADH (or NADPH) -dependent oxidoreductase. Due to the complexity required for cofactors, industrial methods using the reaction sequence for 1,3-propanediol production require the use of whole cell catalysts.
[22] Summary of the Invention
[23] Applicants have addressed the above problem, and the present invention provides a method of directly converting a fermentable carbon source into 1,3-propanediol using a single microorganism, but at a significantly higher titer than previously obtained titers. . Glucose is used as a model substrate and E. coli is used as a model host. In one aspect of the invention, the gene family (dehydratase activity, dehydratase reactivation factor, 1,3-propanediol oxidoreductase (dhaT), glycerol-3-phosphate dehydrogenase, and glycerol-3 Recombinant Escherichia coli expressing (including genes encoding phosphatase) have a titer when converting glucose to 1,3-propanediol to a titer when fermenting glycerol to 1,3-propanediol.
[24] In another aspect of the invention, the removal of the functional dhaT gene from the recombinant E. coli produces a significantly higher titer of 1,3-propanediol in glucose. This unexpected increase in titer improves economics, thus improving the method of producing 1,3-propanediol from glucose.
[25] In addition, the present invention generally relates to the C ₃ compound (eg, glycerol 3-phosphate), or 4) dihydroxyacetone in the oxidized state of 1) glycerol, 2) dihydroxyacetone, 3) the oxidized state of glycerol. It can be applied to include any carbon substrate that is readily converted to C ₃ compounds (eg, dihydroxyacetone phosphate or glyceraldehyde 3-phosphate). Production of 1,3-propanediol in the dhaT ^- strain requires nonspecific catalytic activity of converting 3-HPA to 1,3-propanediol. Identification of enzyme (s) and / or gene (s) responsible for nonspecific catalytic activity of converting 3-HPA to 1,3-propanediol can be achieved by using substrates from a wide range of carbon-containing substrates. In the host microorganism. In addition, using nonspecific catalytic activity of converting 3-HPA to 1,3-propanediol thus improves the method of producing 1,3-propanediol from glycerol or dihydroxyacetone by improving the titer and thus the economics It is expected to be.
[26] The activity was isolated as a nucleic acid fragment encoding a nonspecific catalytic activity that converts 3-hydroxypropionaldehyde to 1,3-propanediol and is set forth in SEQ ID NO: 58 or selected from the group consisting of:
[27] (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO: 57,
[28] (b) an isolated nucleic acid fragment substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO: 57,
[29] (c) an isolated nucleic acid fragment encoding a polypeptide of at least 387 amino acids, having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 57,
[30] (d) Isolated nucleic acid fragments hybridized with (a) under hybridization conditions of 0.1 × SSC, 0.1% SDS, 65 ° C., washed with 2 × SSC, 0.1% SDS and then washed with 0.1 × SSC, 0.1% SDS , And
[31] (e) an isolated nucleic acid fragment complementary to (a), (b), (c), or (d).
[32] Alternatively, nonspecific catalytic activity is implemented in the polypeptide set forth in SEQ ID NO: 57. Chimeric genes can be constructed comprising the isolated nucleic acid fragments described above operably linked to suitable regulatory sequences. Using this chimeric gene, Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter , Lactobacillus, Aspergillus , Saccharomyces, Schizosaccharomyces ), is saccharide as MY access (Zygosaccharomyces), blood teeth (Pichia), Cluj Vero My process (Kluyveromyces), Candida (Candida), Hanse Cronulla (Hansenula), debari Oh, my process (Debaryomyces), non-cor (Mucor), torul rope sheath (Torulopsis), methyl bakteo (Methylobacter), Salmonella, Bacillus, Aero bakteo, Streptomyces (Streptomyces), in Sherry hyacinth (Escherichia), and Pseudomonas (Pseudomonas) can be transformed with the selected micro-organisms from the group consisting of have. Preferred host is E. coli.
[33] Therefore, the present invention relates to (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity, ( c) at least one gene encoding a polypeptide having dehydratase activity, (d) at least one gene encoding a dehydratase reactivation factor, (e) 1,3-propanediol as 3-hydroxypropionaldehyde Recombinant microorganisms useful for producing 1,3-propanediol, comprising one or more endogenous genes that encode sufficient nonspecific catalytic activity to convert to and lacking a functional dhaT gene encoding 1,3-propanediol oxidoreductase To provide. Preferred embodiments are recombinant microorganisms (preferably E. coli) lacking the dhaT gene. If desired, the recombinant microorganism may comprise (a) a gene encoding a polypeptide having glycerol kinase activity, (b) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (c) a triosphosphate isomerase activity Mutations (eg, deletion mutations or point mutations) of endogenous genes selected from the group consisting of genes encoding polypeptides having
[34] In another embodiment, the present invention
[35] (a) Under appropriate conditions, a recombinant E. coli comprising a dha regulator and without a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity, comprises a group consisting of monosaccharides, oligosaccharides, polysaccharides and 1-carbon substrates. Contacting at least one carbon source selected from, and
[36] (b) optionally, a process for producing 1,3-propanediol, comprising recovering 1,3-propanediol produced in step (a).
[37] In addition, the present invention
[38] (a) contacting the recombinant microorganism of the present invention with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and 1-carbon substrates to produce 1,3-propanediol, and
[39] (b) optionally recovering 1,3-propanediol produced in step (a)
[40] It comprises a, 1,3-propanediol production method from a recombinant microorganism.
[41] Similarly, the present invention
[42] (a) at least one gene encoding a polypeptide having dehydratase activity, (ii) at least one gene encoding a dehydratase reactivation factor, (iii) 3-hydroxypropionaldehyde as 1, Recombinant microorganisms comprising at least one endogenous gene encoding a nonspecific catalytic activity sufficient for conversion to 3-propanediol and lacking a functional dhaT gene encoding 1,3-propanediol oxidoreductase are glycerol and dihydroxy Contacting with at least one carbon source selected from the group consisting of acetone to produce 1,3-propanediol, and
[43] (b) optionally recovering 1,3-propanediol produced in step (a)
[44] To provide a method for producing 1,3-propanediol from recombinant microorganisms, including.
[45] Another aspect of the present invention provides a method of simultaneously supplying a carbon substrate. In this embodiment for producing 1,3-propanediol, the steps are
[46] (a) at least one exogenous gene encoding a polypeptide having dehydratase activity, (ii) at least one exogenous gene encoding a dehydratase reactivation factor, and (iii) 3-hydroxypropionaldehyde Recombinant Escherichia coli comprising at least one exogenous gene encoding a nonspecific catalytic activity sufficient to convert to 1,3-propanediol, and lacking a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity, is selected from glycerol and Contacting with a first carbon source selected from the group consisting of dihydroxyacetone and a second carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and 1-carbon substrates, and
[47] (b) optionally recovering 1,3-propanediol produced in step (a). Simultaneous feeding can be sequential or simultaneous. Recombinant Escherichia coli used in the co-feeding embodiment comprises (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, and (ii) a polypeptide having glycerol-3-phosphatase activity. An exogenous gene set consisting of at least one gene encoding and (iii) at least one gene subset encoding gene products of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and (b) (i A gene encoding a polypeptide having glycerol kinase activity, (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (iii) a gene encoding a polypeptide having triosphosphate isomerase activity; , Each gene may further comprise a set of endogenous genes having mutations that inactivate said gene.
[48] Useful recombinant E. coli strains comprise (a) a gene encoding a polypeptide having glycerol kinase activity, and (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, each gene encoding the gene. A set of two endogenous genes having mutations to inactivate, (b) at least one exogenous gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, (c) a polypeptide having glycerol-3-phosphatase activity At least one exogenous gene encoding, and (d) recombinant E. coli strain KLP23 comprising plasmid pKP32; And (a) a gene encoding a polypeptide having glycerol kinase activity, (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (iii) a polypeptide having triosphosphate isomerase activity. Recombinant E. coli strain RJ8, which consists of a gene encoding the gene, and each gene contains a set of three endogenous genes having mutations that inactivate the gene.
[49] Other useful embodiments include (a) at least one gene encoding a polypeptide having dehydratase activity, (ii) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, an exogenous gene set consisting of (iii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity, and (iv) at least one gene encoding a dehydratase reactivation factor, and (b) a 3-hydric Recombinant Escherichia coli comprising at least one endogenous gene encoding a nonspecific catalytic activity for converting oxypropionaldehyde to 1,3-propanediol and lacking a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity Include.
[50] Other embodiments include (a) (i) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, and (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity. And (iii) an exogenous gene set consisting of at least one subset of genes encoding gene products of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and (b) 3-hydroxypropionaldehyde Recombinant E. coli, which comprises at least one endogenous gene encoding a nonspecific catalytic activity of converting to 1,3-propanediol, and lacks a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity. In addition, these embodiments further comprise (a) a gene encoding a polypeptide having glycerol kinase activity, (b) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (c) a triosphosphate isomerase activity It may also comprise a method of using recombinant E. coli, consisting of a gene encoding a polypeptide, each gene comprising a set of endogenous genes having mutations that inactivate said gene.
[51] In addition, these embodiments
[52] (a) under suitable conditions, contacting the recombinant E. coli described above with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and 1-carbon substrates to produce 1,3-propanediol, and
[53] (b) optionally, further providing a method for biological production of 1,3-propanediol, comprising recovering 1,3-propanediol produced in step (a).
[54] Also,
[55] (a) at least one exogenous gene encoding a polypeptide having dehydratase activity, (ii) at least one exogenous gene encoding a dehydratase reactivation factor, and (iii) 3-hydroxypropionaldehyde One or more carbon sources selected from the group consisting of glycerol and dihydroxyacetone, wherein the recombinant E. coli of the above-described embodiment further comprises at least one endogenous gene encoding a nonspecific catalytic activity for conversion to 1,3-propanediol Contacting with an abnormality, and
[56] (b) optionally recovering 1,3-propanediol produced in step (a)
[57] It further comprises a biological production method of 1,3-propanediol comprising a.
[58] BRIEF DESCRIPTION OF THE DRAWINGS, Sequencing and Biological Deposits
[59] The invention can be more fully understood from the following detailed description, figures, and appended sequence descriptions, and from the biological deposits that form part of this specification.
[60] 1 depicts the genetic organization within the dha regulatory subclonal pHK28-26 sequence.
[61] FIG. 2 essentially shows a graph of extracellular soluble protein (g / L) compared between two fermentations run with a constant amount of vitamin B ₁₂ as described in Example 7. FIG. The strain used in the case indicated by the solid line is KLP23 / pAH48 / pKP32. The strain used in the dotted line is KLP23 / pAH48 / pDT29.
[62] FIG. 3 essentially shows a graph of cell viability [(living cell number / mL) / OD ₅₅₀ ] compared between two fermentations run with a constant amount of vitamin B ₁₂ as described in Example 7. . The strain used in the case indicated by the solid line is KLP23 / pAH48 / pKP32. The strain used in the dotted line is KLP23 / pAH48 / pDT29.
[63] FIG. 4 shows a graph of glycerol yield from glucose comparing essentially between two fermentations operated essentially as described in Example 7, but without vitamin B ₁₂ or coenzyme B ₁₂ . The strain used in the case indicated by the solid line is KLP23 / pAH48 / pKP32. The strain used in the dotted line is KLP23 / pAH48 / pDT29.
[64] 5 is a flow chart showing metabolic conversion of glucose to 1,3-propanediol.
[65] FIG. 6 is 2D-PAGE membrane blotting using soluble protein fractions extracted from one band showing endogenous E. coli oxidoreductase activity (nonspecific catalytic activity) on natural gels.
[66] The 68 sequence listings and the sequence listings attached herein are described in 37 C.F.R. As described in §1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures-the Sequence Rules"), in accordance with the provisions for the disclosure of nucleotide and / or amino acid sequences in a patent application; In the World Intellectual Property Organization (WIPO) Standard ST2.5 (1998) and EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administration Instructions) Meets the requirements. Sequence descriptions are given in Nucleic Acids Res. 13, 3021-3030 (1985) and the one letter alphabetic code for the nucleotide sequence and the three letter code for the amino acid, defined in accordance with the IUPAC-IYUB criteria described in Biochemical Journal 219, 345-373 (1984). Which is incorporated herein by reference.
[67] SEQ ID NO: 1 is pKP1 (Cosmid containing DNA of Klebsiella pneumoniae) subcloned with pIBI31 (IBI Biosystem, New Haven, Connecticut) (referred to as pHK28-26) ) Contains the nucleotide sequence determined from the 12.1 kb EcoRI-SalI fragment. Table 1 describes in more detail the genes, corresponding base pairs identified in SEQ ID NO: 1, and related functions. See Example 1.
[68] SEQ ID NO: 57 contains the nucleotide sequence determined for yqhD.
[69] Applicants have biologically deposited the following under the Budapest Treaty on International Approval of Microbial Deposits in Patent Procedures.
[70] SEQ ID NO: 58 contains an amino acid sequence determined for yqhD.
[71] Identification mark given by the depositary International accession number Date of entrustment Transformed Escherichia coli DH5α containing a portion of the Klebsiella genome encoding the glycerol dehydratase enzyme ATCC 69789 April 18, 1995 Transformed Escherichia coli DH5α containing a portion of the Klebsiella genome encoding a diol dehydratase enzyme ATCC 69790 April 18, 1995 Escherichia coli MSP33.6 ATCC 98598 November 25, 1997 glpK mutant Escherichia coli RJF 1Om ATCC 98597 November 25, 1997
[72] The deposit (s) will be held for more than 30 years at the designated international depositary institution and will be made publicly available as soon as the patent which discloses it is approved. In infringement of patent rights granted by administrative activities, the availability of such deposits does not constitute a license for practicing the present invention.
[73] As used herein, “ATCC” refers to the American Type Culture Collection International Depository, Manilas University Boulevard 10801, 20110-2209, USA. "ATCC Number" is the accession number after depositing the culture with the ATCC.
[74] Detailed description of the invention
[75] The present invention provides an improved method for the direct bioconversion of fermentable carbon sources to 1,3-propanediol using a single microorganism. The method is characterized by reduced cell lysis during fermentation as well as improvement in titer, yield, and cell viability.
[76] The present invention is directed to a process in which 1,3-propanediol fermentation, including 1,3-propanediol oxidoreductase (dhaT), correlates with reduced cell viability in the medium, It is based on the observation that there is a characteristic of high concentration. In addition, the present invention is directed, in part, to the model host E. coli to convert 3-HPA to 1,3-propanediol by endogenous nonspecific catalytic activity capable of converting 3-hydroxypropionaldehyde to 1,3-propanediol. It is based on an unexpected discovery that it can. In addition, the present invention provides that, in part, the E. coli fermentation process that includes this non-specific catalytic activity and no functional dhaT increases cell viability during fermentation, the titer of 1,3-propanediol over the fermentation process comprising functional dhaT and ( Or) based on unexpected findings of high yields.
[77] In one aspect, the model substrate is glycerol, the host microorganism is mutated in wild type dhaT and lacks 1,3-propanediol oxidoreductase activity and converts 3-hydroxypropionaldehyde to 1,3-propanediol Sufficient non-specific catalytic activity. In another aspect, the model substrate is glucose and the model host is recombinant E. coli. In this aspect, Escherichia coli comprises endogenous nonspecific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol. In one embodiment, said nonspecific catalytic activity is alcohol dehydrogenase.
[78] In one aspect, the invention provides an antibody comprising (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, and (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity. (c) at least one gene encoding a polypeptide having dehydratase activity, (d) at least one gene encoding a dehydratase reactivation factor, and (e) 3-hydroxypropionaldehyde as a 1,3 Providing a recombinant E. coli expressing a group of genes comprising at least one endogenous gene encoding a nonspecific catalytic activity sufficient for conversion to propanediol; The microorganism is used to convert glucose to 1,3-propanediol at high titers. In another aspect of the invention, removing the functional dhaT gene from the recombinant E. coli provides a titer from glucose to 1,3-propanediol at an unexpectedly higher titer than previously achieved.
[79] The present invention provides an improved method for biologically producing 1,3-propanediol from a carbon source fermentable in a single microorganism. In one aspect of the invention, improved methods for converting glucose to 1,3-propanediol include the Klebsiella pneumoniae dha regulatory genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, And recombinant microorganisms comprising host E. coli transformed with orfZ (all of these genes are arranged in the same genetic tissue as found in wild type Klebsiella pneumoniae). The titer obtained by the fermentation method is considerably higher than the potency previously reported for similar fermentation methods. This improvement depends on the use of plasmid pDT29, as described in Examples 6 and 7.
[80] In another aspect of the invention, a method of using recombinant E. coli containing genes encoding G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factor to produce 1,3-propanediol from glucose comprises It is an improvement over the use of recombinant E. coli, which contains not only G3P phosphatase, dehydratase, dehydratase reactivation factor, but also functional dhaT. The dramatic improvement of this method depends on the presence of endogenous genes encoding nonspecific catalytic activity predicted by alcohol dehydrogenase present in E. coli.
[81] As described in Examples 7 and 9, a dramatic improvement in the process is evident with increasing 1,3-propanediol titers. In addition, the improvement in this method is also evident by the decrease in cell lysis rate, determined by the concentration of extracellular soluble protein in the fermentation broth. This aspect of the invention is described in FIG. 2. In addition, improvements in this method are also evident in the extension of cell viability throughout the fermentation process. This aspect of the invention is described in FIG. 3. In addition, the improvement in the process is also evident in the increase in yield. In E. coli expressing 1,3-propanediol oxidoreductase (dhaT) (eg, E. coli KLP23 transformed with plasmid pDT29), glycerol can be metabolized to a product other than 3-HPA. On the contrary, in E. coli that does not express 1,3-propanediol oxidoreductase (dhaT) (eg, E. coli KLP23 transformed with plasmid pKP32), glycerol is not metabolized to a product other than 3-HPA. It was demonstrated that this cryptic pathway can be attributed to the presence or absence of functional dhaT, as shown in FIG. 4, with reduced glycerol yield from glucose.
[82] As used herein, the following terms may be used to understand the claims and the specification.
[83] The terms "glycerol-3-phosphate dehydrogenase" and "G3PDH" refer to polypeptides responsible for enzymatic activity catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). In vivo G3PDH may be NADH-, NADPH-, or FAD-dependent. In particular, when referring to cofactors specific for glycerol-3-phosphate dehydrogenase, the terms "NADH-dependent glycerol-3-phosphate dehydrogenase", "NADPH-dependent glycerol-3-phosphate dehydrogenase" And "FAD-dependent glycerol-3-phosphate dehydrogenase" will be used. As is common when NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenases can use NADH and NADPH interchangeably (for example, by genes that gpsA encodes), the term NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenases will be used interchangeably. NADH-dependent enzyme (EC 1.1.1.8) is encoded by several genes, such as GPD1 (Genbank Z74071x2), or GPD2 (Genbank Z35169x1), or GPD3 (Genbank G984182), or DAR1 (Genbank Z74071x2). NADPH-dependent enzymes (EC 1.1.1.94) are encoded by gpsA (Genbank U321643, (cds 197911-196892) G466746 and L45246). FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2 (Genbank Z47047x23), or glpD (Genbank G147838), or glpABC (Genbank M20938) (WO 9928480 and references herein incorporated by reference) Reference).
[84] The terms "glycerol-3-phosphatase", "sn-glycerol-3-phosphatase", or "d, l-glycerol phosphatase", and "G3P phosphatase" mean the conversion of glycerol-3-phosphate and water to glycerol and inorganic phosphate It refers to a polypeptide that is responsible for enzymatic activity that catalyzes. G3P phosphatase is encoded by GPP1 (Genbank Z47047x125), or GPP2 (Genbank U18813x11) and the like (see WO 9928480 and references herein incorporated by reference).
[85] The term "glycerol kinase" refers to a polypeptide that is responsible for enzymatic activity that catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP. ATP, a high energy phosphate donor, can be replaced with a physiological substituent (eg, phosphoenolpyruvate). Glycerol kinases are encoded by GUT1 (Genbank U11583x19) and glpK (Genbank L19201) and the like (see WO 9928480 and references therein, incorporated herein by reference).
[86] The term "glycerol dehydrogenase" means enzymatic activity catalyzing the conversion of glycerol to dihydroxyacetone (EC1.1.1.6) or enzymatic activity catalyzing the conversion of glycerol to glyceraldehyde (EC1.1.1.72). Refers to a polypeptide responsible for. Polypeptides responsible for enzymatic activity catalyzing the conversion of glycerol to dihydroxyacetone are also referred to as "dihydroxyacetone reductases". Glycerol dehydrogenase may be NADH-dependent (E.C.1.1.1.6), NADPH-dependent (E.C.1.1.1.72), or other cofactor-dependent (eg, E.C.1.1.99.22). NADH-dependent glycerol dehydrogenase is encoded by gldA (Genbank U00006) et al. (See WO 9928480 and references therein, incorporated herein by reference).
[87] The term "dehydratase enzyme" or "dehydratase" refers to any enzymatic activity that catalyzes the conversion of the product 3-hydroxypropionaldehyde of the glycerol molecule. For the purposes of the present invention, the dehydratase enzyme comprises glycerol dehydratase (EC4.2.1.30) and diol dehydratase (EC4.2.1.28) (preferred substrates are glycerol and 1,2-propanediol, respectively) . Genes for dehydratase enzymes have been identified in Klebsiella pneumoniae, Citrobacter proindei, Clostridium pasteerianum, Salmonella typhimurium, and Klebsiella oxytoca. In each case, the dehydratase consists of three subunits: a large subunit or "α" subunit, an intermediate subunit or "β" subunit, and a small subunit or "γ" subunit. Since the gene nomenclature used in the literature varies, a comparative chart is provided in Table 1 for ease of identification. In addition, the genes are described, for example, in Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999) and Toraya and Mori, J Biol. Chem. 274, 3372 (1999) and the like. Referring to Table 1, genes encoding a large subunit or "α" subunit of glycerol dehydratase include dhaB1, gldA and dhaB, and the like, and genes encoding an intermediate subunit or "β" subunit. dhaB2, gldB, and dhaC, and the like, and genes encoding small subunits or “γ” subunits include dhaB3, gldC, and dhaE. Referring back to Table 1, genes encoding a large subunit or "α" subunit of diol dehydratase include pduC and pddA, and the like, and genes encoding an intermediate subunit or "β" subunit are pduD. And pddB and the like, and genes encoding small subunits or “γ” subunits include pduE and pddC.
[88] Gene Names and Genbank Reference Numbers for Dehydratase, and Comparative Charts for Dehydratase Related Functions Gene function control Unknown Reactivation 1,3-PD dehydrogenase Unknown gene Base pair gene Base pair gene Base pair gene Base pair gene Base pair Organisms (Genbank Reference Number) Klebsiella pneumoniae (SEQ ID NO: 1) dhaR 2209-4134 orfW 4112-4642 orfX 4643-4996 dhaT 5017-6108 orfY 6202-6630 Klebsiella pneumoniae (U30903)orf2c 7116-7646 orf2b 6762-7115 dhaT 5578-6741 orf2a 5125-5556 Klebsiella pneumoniae (u60992) gdrB Citrobacter Prodydy (U09771) dhaR 3746-5671 orfW 5649-6179 orfX 6180-6533 dhaT 6550-7713 orfY 7736-8164 Clostridium Pasterianum (AF051373) Clostridium Pasterianum (AF006034)orfW 210-731 orfX 1-196 dhaT 1232-2389 orfY 746-1177 Salmonella typhimurium (AF026270) pduH 8274-8645 Klebsiella oxytoca (AF017781) ddrB 2063-2440 Klebsiella oxytoca (AF051373)
[89] Gene function Dehydratase, α Dehydratase, β Dehydratase, γ Reactivation gene Base pair gene Base pair gene Base pair gene Base pair Organisms (Genbank Reference Number) Klebsiella pneumoniae (SEQ ID NO: 1) dhaB1 7044-8711 dhaB2 8724-9308 dhaB3 9311-9736 orfZ 9749-11572 Klebsiella pneumoniae (U30903) dhaB1 3047-4714 dhaB2 2450-2890 dhaB3 2022-2447 dhaB4 186-2009 Klebsiella pneumoniae (u60992) gldA 121-1788 gldB 1801-2385 gldC 2388-2813 gdrA Citrobacter Prodydy (U09771) dhaB 8556-10223 dhaC 10235-10819 dhaE 10822-11250 orfZ 11261-13072 Clostridium Pasterianum (AF051373) dhaB 84-1748 dhaC 1779-2318 dhaE 2333-2773 orfZ 2790-4598 Clostridium Pasterianum (AF006034) Salmonella typhimurium (AF026270) pduC 3557-5221 pduD 5232-5906 pduE 5921-6442 pduG 6452-8284 Klebsiella oxytoca (AF017781)ddrA 241-2073 Klebsiella oxytoca (AF051373) pddA 121-1785 pddB 1796-2470 pddC 2485-3006
[90] Glycerol and diol dehydratase are subject to suicide inactivation based on mechanisms by glycerol and some other substrates [Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999). The term "dehydratase reactivation factor" refers to proteins responsible for reactivation of dehydratase activity. The terms "dehydratase reactivation activity", "reactivating dehydratase activity" or "reproducing dehydratase activity" mean that a dehydratase capable of catalyzing a substrate is capable of catalyzing a substrate. Conversion to other drugs, inhibition of dehydratase inactivation, or extension of useful half-life of dehydratase enzymes in vivo. Two proteins associated with dehydratase reactivation factors have been identified [WO 9821341 (US Pat. No. 6013494) and references therein, Daniel et al., Supra, Toraya and Mori, J. Biol. Chem. 274, 3372 (1999) and Tobimatsu et al., J. Bacteriol. 181, 4110 (1999), which is incorporated herein by reference). Referring to Table 1, genes encoding one of the two proteins include orfZ, dhaB4, gdrA, pduG, and ddrA. In addition, referring to Table 1, genes encoding the other of the above are orfX, org2b, gdrB, pduH and ddrB.
[91] The terms "1,3-propanediol oxidoreductase", "1,3-propanediol dehydrogenase" or "DhaT" mean that the gene (s) encoding the activity is in its natural form (ie, wild type) Refers to polypeptide (s) responsible for enzymatic activity capable of catalyzing the interconversion of 3-HPA and 1,3-propanediol, found to be physically or transcriptionally related to the dehydratase enzyme; For example, in the case of dhaT of Klebsiella pneumoniae, this gene is found in the dha regulator. Referring to Table 1, genes encoding 1,3-propanediol oxidoreductase include Klebsiella pneumoniae, Citrobacter proindy, and dhaT of Clostridium pasteerianum. Each of these genes encodes polypeptides belonging to the type III alcohol dehydrogenase family, iron-binding motifs are conserved, and preferentially to the NAD ⁺ / NADH related interconversions of 3-HPA and 1,3-propanediol (Johnson and Lin, J. Bacteriol. 169, 2050 (1987)), Daniel et al., J. Bacteriol. 177, 2151 (1995), and Leurs et al., FEMS Microbiol. Lett. 154, 337 (1997)]. Enzymes with similar physical properties are described in Veiga da Dunha and Foster, Appl. Environ. Microbiol. 58, 2005 (1992).
[92] The term "dha regulator" refers to an open reading frame or a set of related genes encoding various biological activities, which include dehydratase activity, reactivation activity, 1,3-propanediol oxidoreductase, and the like. However, it is not limited thereto. Typically, as described herein, dha regulators include open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ.
[93] The term "nonspecific catalytic activity" refers to polypeptide (s) responsible for sufficient enzymatic activity to catalyze the interconversion of 3-HPA and 1,3-propanediol, specifically 1,3-propanediol oxidoreductase (S) is excluded. Typically, these enzymes are alcohol dehydrogenases. The enzymes may use cofactors other than NAD ⁺ / NADH, but such cofactors include, but are not limited to, flavin such as FAD or FMN. Gene (s) relating to nonspecific alcohol dehydrogenase (s) have been found to be endogenously encoded and functionally expressed, for example, in microbial Escherichia coli KLP23. .
[94] The term "function" or "enzyme function" refers to the catalytic activity of an enzyme with altered energy required to perform a specific chemical reaction. It is understood that this activity can be applied to equilibrium reactions where product or substrate production can be carried out under suitable conditions.
[95] The terms "polypeptide" and "protein" are used interchangeably.
[96] The terms "carbon substrate" and "carbon source" are carbon sources selected from the group consisting of carbon sources that can be metabolized by the host microorganisms of the invention, in particular monosaccharides, oligosaccharides, polysaccharides, and 1-carbon substrates or mixtures thereof. Refers to the source.
[97] The term "host cell" or "host microorganism" refers to a microorganism capable of receiving foreign or heterologous genes and expressing these genes to produce active gene products.
[98] The terms “foreign gene”, “foreign DNA”, “heterologous gene” and “heterologous DNA” refer to the genetic material in which a natural genetic material in some microorganisms is placed in the host microorganism by various means. The gene of interest may be a naturally occurring gene, a mutated gene, or a synthetic gene.
[99] The terms "transformation" and "transfection" refer to the acquisition of a new gene in a cell after introduction of the nucleic acid. The gene obtained can be integrated into chromosomal DNA or introduced as an extrachromosomal replication sequence. The term "transformer" refers to the product of transformation.
[100] The term "genetically altered" refers to the process of changing a genetic material by transformation or mutation.
[101] The terms “recombinant microorganism” and “transformed host” refer to any microorganism transformed with a heterologous or foreign gene, or an extra copy of a homologous gene. Recombinant microorganisms of the invention are glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2) for producing 1,3-propanediol from suitable carbon substrates. And dhaB3), dehydratase reactivation factors (orfZ and orfX), and optionally a foreign gene encoding 1,3-propanediol oxidoreductase (dhaT). Preferred embodiments are E. coli transformed with the gene but without functional dhaT. In addition, host microorganisms other than Escherichia coli contain the genes disclosed above and genes related to non-specific catalytic activity for interconversion of 3-HPA and 1,3-propanediol, but in particular 1,3-propanediol oxidoreductase ( Genes (dhaT) can be transformed to exclude.
[102] A "gene" refers to a nucleic acid fragment that expresses a specific protein, including the front (5 'noncoding) and back (3' noncoding) regulatory sequences of the coding region. The terms "natural" and "wild-type" refer to genes that exist in nature and their native regulatory sequences.
[103] The terms "encoding" and "coding" refer to the process by which a gene produces amino acid sequences through transcription and translation mechanisms. The process of encoding a particular amino acid sequence can include base changes that do not result in a change in the amino acid being encoded, or bases that can change one or more amino acids but do not affect the functional properties of the protein encoded by the DNA sequence. It is understood to include change. Therefore, it is to be understood that the present invention encompasses more than specifically exemplified sequences.
[104] The term "isolated" refers to a protein or DNA sequence from which one or more naturally bound components have been removed.
[105] An “isolated nucleic acid molecule” refers to a polymer of single or double stranded DNA or RNA, which may optionally include synthetic, unnatural or modified nucleotide bases. Isolated nucleic acid molecules in the form of DNA polymers may comprise one or more fragments of cDNA, genomic DNA or synthetic DNA.
[106] “Substantially similar” refers to a nucleic acid molecule in which one or more nucleotide base changes can substitute for one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to a nucleic acid molecule in which one or more nucleotide base changes do not affect the ability of the nucleic acid molecule to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also substantially affects the functional properties of the resulting transcript when compared to the ability to mediate alteration of gene expression by antisense or co-inhibition techniques, or alteration of the functional properties of the resulting protein molecules. Refers to modifications of nucleic acid molecules of the invention, such as one or more nucleotide base deletions or insertions, that do not extend. The present invention specifically encompasses more than illustrated sequences.
[107] For example, it is known in the art that altering a gene produces chemically equivalent amino acids at that site and does not affect the functional properties of the protein being encoded. For the purposes of the present invention, substitutions are defined as exchanges in one of the following five groups:
[108] 1. small aliphatic, nonpolar residues or small aliphatic, somewhat polar residues: Ala, Ser, Thr (Pro, Gly),
[109] 2. Polar, negatively charged residues and amides thereof: Asp, Asn, Glu, Gln,
[110] 3. Polar, positively charged residues: His, Arg, Lys,
[111] 4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys), and
[112] 5. Large aromatic residues: Phe, Tyr, Trp.
[113] Therefore, the codons of alanine, a hydrophobic amino acid, can be substituted with codons encoding other less hydrophobic residues (eg glycine) or more hydrophobic residues (eg valine, leucine, or isoleucine). . Similarly, substitution of one negatively charged residue with another negatively charged residue (eg, aspartic acid with glutamic acid), or one positively charged residue with another positively charged residue ( For example, changes in lysine substitution with arginine may also be expected to produce functionally equivalent products.
[114] In many cases, it is predicted that nucleotide changes that alter the N- and C-terminal portions of a protein molecule will not alter the protein's activity.
[115] Each of the proposed modifications is within the ordinary skill in the art, such as to determine whether the encoded product retains biological activity. Also, those skilled in the art will recognize that substantially similar sequences included in the present invention also hybridize with the sequences exemplified herein under stringent conditions (0.1 × SSC, 0.1% SDS, 65 ° C., and then washed with 2 × SSC, 0.1% SDS). It will be understood that this is defined by the ability to hybridize under 0.1 × SSC, wash with 0.1% SDS). Substantially similar nucleic acid fragments preferred for the present invention are nucleic acid fragments having at least 80% DNA sequence identity with nucleic acid fragments as reported herein. More preferred nucleic acid fragments are nucleic acid fragments having at least 90% DNA sequence identity with nucleic acid fragments as reported herein. Most preferred are nucleic acid fragments having at least 95% DNA sequence identity with nucleic acid fragments as reported herein.
[116] If the single stranded form of the nucleic acid fragment can be annealed with other nucleic acid fragments such as cDNA, genomic DNA, or RNA under conditions of moderate temperature and ionic strength of the solution, the nucleic acid fragment is “hybridizable” with the other nucleic acid fragment. . Hybridization and washing conditions are known and are described in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), in particular the above. Chapter 11 of the literature and Table 11.1, which are hereby incorporated by reference in their entirety. The conditions of temperature and ionic strength determine the "stringency" of hybridization. Preliminary screening for homologous nucleic acids includes low stringency hybridization conditions with a Tm of 55 ° C., such as 5 × SSC, 0.1% SDS, 0.25% milk, and no formamide; Or conditions with 30% formamide, 5 x SSC, 0.5% SDS can be used. Moderate stringency hybridization conditions correspond to higher temperature Tm, for example 40% formamide, 5 × or 6 × SSC. Hybridization requires that two nucleic acids contain complementary sequences, but mismatches between bases are possible depending on the stringency of the hybridization. Suitable stringency for nucleic acid hybridization depends on the length of the nucleic acid and the degree of complementarity which are variables known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the Tm value for the hybrid of the nucleic acid having that sequence. Relative stability of nucleic acid hybridization (corresponding to high temperature Tm) decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. In hybrids greater than 100 nucleotides in length, formulas for calculating Tm can be derived from Sambrook et al., Supra, 9.50-9.51. For hybridization with shorter nucleic acids, ie oligonucleotides, the location of the mismatch becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8). . In one embodiment, the hybridizable nucleic acid is at least about 10 nucleotides in length. The preferred minimum length of the hybridizable nucleic acid is at least about 15 nucleotides, more preferably at least about 20 nucleotides, most preferably at least 30 nucleotides in length. Those skilled in the art will also appreciate that the temperature and salt concentration of the wash solution can be adjusted as needed depending on factors such as the length of the probe and the like.
[117] By "corresponding moiety", one skilled in the art can manually assess the sequence, or use BLAST (Basic Local Alignment Search Tool, Altschul et al., J. Mol. Biol. 215: 403-410 (1993)), and also www.ncbi. automated computer sequence comparison and identification using algorithms such as .nlm.nih.gov / BLAST /) to include the amino acid sequence of a polypeptide or the nucleotide sequence of a gene sufficient to infer about the polypeptide or its genes. Refers to an amino acid or nucleotide sequence. Typically, 10 or more contiguous amino acid sequences or 30 or more nucleotide sequences are required to estimate and identify any polypeptide or nucleic acid sequence as a homologue for a known protein or gene. In addition, for nucleotide sequences, gene-specific oligonucleotide probes, including 20-30 consecutive nucleotides, can be sequence-dependent gene identification (eg, Southern hybridization) and isolation (eg, bacterial colonies or bacteriophages). In situ hybridization of plaques). In addition, short oligonucleotides of 12-15 bases can be used as amplification primers in PCR to obtain specific nucleic acid molecules comprising the primers. Therefore, a “significant portion” of a nucleotide sequence includes a sequence sufficient for the specific identification and / or isolation of the nucleic acid molecule comprising the sequence. The present specification teaches nucleotide sequences encoding partial or complete amino acids and one or more specific proteins. One skilled in the art, using the advantages of such sequences as reported herein, will be able to use all or a substantial portion of the disclosed sequences for purposes known to those skilled in the art. Therefore, the present invention includes not only the complete sequence as reported in the appended sequence listing, but also a substantial portion of that sequence as defined above.
[118] The term "complementary" refers to a relationship between nucleotide bases that can hybridize to each other. For example, in the case of DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Therefore, the present invention also includes isolated nucleic acid molecules complementary to their substantially similar nucleic acid sequences as well as the complete sequence as reported in the appended sequence listing.
[119] As is known in the art, the term "% identity" is the relationship between the sequences, determined by comparing two or more polypeptide sequences or two or more polynucleotide sequences. In the art, “identity” also means the degree of sequence relevance between the sequences, determined by a match between the polypeptide sequences or a string of polynucleotide sequences. "Identity" and "similarity" can be easily calculated by known methods, which include Computational Molecular Biology; Lesk, A. M., Ed., Oxford University Press: New York, 1988, Biocomputing: Informatics and Genome Projects; Smith, D. W., Ed .; Academic Press: New York, 1993, Computer Analysis of Sequence Data, Part 1; Griffin, A. M. and Griffin, H. G., Eds., Humana Press: New Jersey, 1994], Sequence Analysis in Molecular Biology; von Heinje, G., Ed., Academic Press: New York, 1987, and Sequence Analysis Primer; Gribskov, M. and Devereux, J., Eds., Stockton Press: New York, 1991, and the like. Preferred methods for determining identity are designed to provide the largest match between the sequences tested.
[120] Methods of measuring identity and similarity are documented in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include the GCG program package using Needleman and Wunsch algorithms using standard default values with gap generation penalty = 12 and gap extension penalty = 4. GCG pile-up program (Devereux et al., Nucleic Acids Res. 12: 387-395 (1984)), BLASTP, BLASTN, and FASTA [Pearson et al., Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988)] The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. Library). Med. (NCBI NLM) NIH, Bethesda, Md. 20894, Altschul et al., J. Mol. Biol. 215: 403-410 (1990), Altschul et al., “Gapped BLAST and PSI-BLAST : a new generation of protein database search programs ", Nucleic Acids Res. 25: 3389-3402 (1997)]). Other preferred measures of percent identity. The method is by the DNASTAR protein alignment protocol method using the Jotun-Hein algorithm (Hein et al., Methods Enzymol. 183: 626-645 (1990)) The default of the Joton-Hein method for alignment. The parameters are as follows: for multiple alignments, gap penalty = 11, gap length penalty = 3; for pairwise alignment, ktuple = 6. To explain, the reference nucleotide sequence has, for example, at least 95% "identity"; For a polynucleotide having a nucleotide sequence, the nucleotide sequence of that polynucleotide is the same as the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations for each 100 nucleotides of the reference nucleotide sequence. It means. That is, in order to obtain a polynucleotide having a nucleotide sequence having 95% or more of sequence identity with a reference nucleotide sequence, 5% or less of the nucleotides of the reference sequence may be deleted or replaced with another nucleotide, or the whole of the reference sequence may be removed. Many nucleotides up to 5% of the nucleotides can be inserted into the reference sequence. Such reference sequence mutations may occur at the 5 'end or 3' end position of the reference nucleotide sequence, or occur at any point between the two terminal positions, and may be interspersed separately between nucleotides in the reference sequence or in one or more consecutive groups of the reference sequence. Can be. Similarly, for a polypeptide having an amino acid sequence having, for example, at least 95% "identity" in a reference amino acid sequence, the amino acid sequence of that polypeptide will comprise no more than 5 amino acid changes for each 100 amino acids of the reference amino acid. Except that it can mean that the amino acid sequence of the polypeptide is identical to the reference sequence. That is, in order to obtain a polypeptide having an amino acid sequence having 95% or more of sequence identity with a reference amino acid sequence, 5% or less of the amino acid residues of the reference sequence can be deleted or replaced with another amino acid, or the whole of the reference sequence can be obtained. Many amino acids up to 5% of the amino acid residues can be inserted into the reference sequence. Such alteration of the reference sequence may occur at the amino or carboxy terminal position of the reference amino acid sequence, or may occur at any point between the two terminal positions, and may be interspersed separately between residues in the reference sequence or in one or more consecutive groups of the reference sequence. .
[121] The term “homology” refers to a protein or polypeptide that is naturally occurring or natural in a given host cell. The present invention includes microorganisms that produce homologous proteins via recombinant DNA technology.
[122] The term "% homology" refers to the degree of amino acid sequence identity between polypeptides. If the first amino acid sequence is the same as the second amino acid sequence, the first and second amino acid sequences show 100% homology. Homology between any two polypeptides is directly proportional to the total number of amino acids matched at a given position in either sequence, e.g., if half of the total number of amino acids in either sequence are equal, The two sequences are said to exhibit 50% homology.
[123] "Codon degeneracy" refers to the diversity of genetic code that allows for variations in the nucleotide sequence without affecting the amino acid sequence of the encoded polypeptide. Therefore, the present invention relates to any nucleic acid molecule encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO: 57. Those skilled in the art will know the "codon-bias" exhibited by a particular host cell when using nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene such that expression in the host cell is improved, it is desirable to design the gene such that the frequency of codon use is appropriate for the desired codon usage of that host cell.
[124] Sequence modifications, such as deletions, insertions, or substitutions in sequences, that result in silent changes that do not substantially affect the functional properties of the resulting protein molecules are also contemplated. For example, alterations in the gene sequence to reflect synonymity of the genetic code or to produce chemically equivalent amino acids at a given site are contemplated. Thus, the hydrophobic amino acid alanine codon is replaced with a codon encoding another less hydrophobic residue (eg glycine), or a codon encoding a more hydrophobic residue (eg valine, leucine, or isoleucine). You can. Similarly, a change in replacing a negatively charged cupper with another negatively charged residue (eg, aspartic acid with glutamic acid), or a positively charged residue with another positively charged residue (eg For example, the substitution of lysine with arginine is also expected to produce a biologically equivalent product. Nucleotide changes that alter the N-terminal and C-terminal portions of a protein molecule are also predicted not to alter the activity of that protein. In fact, in some cases it may be desirable to make mutants of the sequence to study the effect on altering the biological activity of the protein. Each of the proposed modifications is within the ordinary skill in the art, such as to determine whether the encoded product retains biological activity. Those skilled in the art will also appreciate that the sequences included in the present invention are also defined by the ability to hybridize with the sequences exemplified herein under stringent conditions (0.1 × SSC, 0.1% SDS, 65 ° C.).
[125] The term "expression" refers to the transcription and translation from a gene encoding a sequence of a gene product into the gene product.
[126] The terms "plasmid", "vector", and "cassette" refer to extra chromosomal elements that often contain genes that are not part of the central metabolism of a cell and are usually in the form of circular double stranded DNA molecules. The elements can be autonomous replication sequences, genomic integration sequences, phage or nucleotide sequences, which are linear or circular of single- or double-stranded DNA or RNA derived from any source, with many of the nucleotide sequences therein for the selected gene product. Promoter fragments and DNA sequences are linked or recombined into unique structures that can be introduced into cells with suitable 3 ′ untranslated sequences. A "transformation cassette" refers to a particular vector that contains a foreign gene and includes not only the foreign gene but also elements that facilitate the transformation of a particular host cell. An "expression cassette" refers to a particular vector that contains a foreign gene and includes not only the foreign gene but also elements that enhance the expression of the gene in a foreign host.
[127] Construction of Recombinant Organisms
[128] Recombinant organisms containing the necessary genes encoding the enzymatic pathway to convert the carbon substrate to 1,3-propanediol can be constructed using techniques known in the art. Glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2, and dhaB3), dehydratase reactivation factors (orfZ and orfX) and 1,3 Genes encoding propanediol oxidoreductase (dhaT) were isolated from natural hosts such as Klebsiella or Saccharomyces and used to transform host strains such as E. coli DH5α, ECL707, AA200, or KLP23. I was.
[129] Isolation of genes
[130] Methods of obtaining the desired gene from the bacterial genome are conventional and are known in the field of molecular biology. For example, if the gene sequence is known, suitable endonuclease cleavage can be used to construct a suitable genomic library and screened using probes complementary to the desired gene sequence. Once the sequence is isolated, a suitable amount of DNA for transformation can be amplified by amplification of the DNA using amplification methods directed to standard primers such as polymerase chain reaction (PCR) [US Pat. No. 4,683,202], and the like. Can be obtained.
[131] Alternatively, cosmid libraries can be constructed where large fragments of genomic DNA (35-45 kb) can be packaged into a vector and used to transform the appropriate host. Cosmid vectors are the only vectors that can accommodate large amounts of DNA. Typically, cosmid vectors contain at least one copy of the cos DNA sequence required for packaging and subsequent cyclization of foreign DNA. In addition to the cos sequence, the vector will also contain origins of replication such as ColE1 and the like, and drug resistance markers such as ampicillin or neomycin resistance genes. Methods of using cosmid vectors to transform suitable bacterial hosts are described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, The literature is incorporated herein by reference.
[132] Typically, to clone the cosmid, foreign DNA is isolated and ligated adjacent to the cos region of the cosmid vector using appropriate restriction endonucleases. The cosmid vector containing the linearized foreign DNA is then reacted with a DNA packaging carrier such as bacteriophage. During the packaging process, the cos site is cut so that the foreign DNA is packaged into the head portion of the viral particles of the bacteria. This particle is then used to transfect suitable host cells, such as E. coli. Once injected into the cell, the foreign DNA is circularized under the influence of the cos sticky end. In this way, large segments of foreign DNA can be introduced into and expressed in recombinant host cells.
[133] Isolation and cloning of genes encoding glycerol dehydratase (dhaB1 dhaB2, and dhaB3), dehydratase reactivation factors (orfZ and orfX), and 1,3-propanediol dehydrogenase (dhaT)
[134] In accordance with the present invention, cosmid vectors and cosmid transformation methods were used to clone large segments of genomic DNA of the genus of bacteria known to contain genes capable of processing glycerol into 1,3-propanediol. . Specifically, the genomic DNA of Klebsiella pneumoniae was isolated by methods known in the art, inserted into the cosmid vector Supercos 1 using the restriction enzyme Sau3A, and packaged using the GigapackII packaging extract. Vector E. coli XL1-Blue MR cells were prepared and transformed with cosmid DNA. Cells were grown in the presence of glycerol and the medium was screened for the ability to convert glycerol to 1,3-propanediol by analyzing the medium for 1,3-propanediol formation.
[135] Two 1,3-propanediol positive transformants were analyzed and the cosmids were named pKP1 and pKP2. DNA sequencing revealed extensive homology to the glycerol dehydratase gene of Citrobacter proindei, which demonstrated that the transformants contained DNA encoding the glycerol dehydratase gene. Other 1,3-propanediol positive transformants were analyzed and their cosmids were named pKP4 and pKP5. DNA sequencing revealed that this cosmid contains DNA encoding the diol dehydratase gene.
[136] In the present invention, a gene isolated from Klebsiella cosmid is used, but other sources of dehydratase gene and dehydratase reactivation factor gene include, but are not limited to, citrobacter, clostridium, and salmonella. (See Table 1).
[137] Genes encoding G3PDH and G3P phosphatase
[138] The present invention provides genes suitable for expressing G3PDH and G3P phosphatase activity in host cells.
[139] Genes encoding G3PDH are known. For example, GPD1 was isolated from Saccharomyces and its base sequence encoding the amino acid sequence of SEQ ID NO: 54 is set forth in SEQ ID NO: 53 (Wang et al., Supra). Similarly, G3PDH activity encoded by GPD2 was also isolated in Saccharomyces [Eriksson et al., Mol. Microbiol. 17, 95 (1995).
[140] For the purposes of the present invention, any gene encoding a polypeptide responsible for NADH-dependent G3PDH activity capable of catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) is considered suitable. . In addition, any gene encoding the amino acid sequence of NADH-dependent G3PDH corresponding to genes DAR1, GPD1, GPD2, GPD3, and gpsA will be functional in the present invention, which amino acid sequence does not alter the function of the enzyme. It is contemplated that this may include deletions or additions. Those skilled in the art will appreciate that genes encoding G3PDH isolated from other sources are also suitable for use in the present invention. Genes encoding G3P phosphatase are known. For example, GPP2 was isolated from Saccharomyces cerevisiae and its base sequence encoding the amino acid sequence given in SEQ ID NO: 56 is set forth in SEQ ID NO: 55 [Norbeck et al., J. Biol. Chem. 271, 13875 (1996).
[141] For the purposes of the present invention, any gene encoding G3P phosphatase activity, which can catalyze the conversion of glycerol-3-phosphate + H ₂ 0 to glycerol + inorganic phosphate, is considered suitable for use in the method. In addition, any gene encoding the amino acid sequence of G3P phosphatase corresponding to genes GPP2 and GPP1 will be functional in the present invention, which amino acid sequence will include amino acid substitutions, deletions or additions that do not alter the function of the G3P phosphatase enzyme. It is considered possible. Those skilled in the art will appreciate that genes encoding G3P phosphatase isolated from other sources are also suitable for use in the present invention.
[142] Host cell
[143] Suitable host cells for recombinant production of 1,3-propanediol may be prokaryotic or eukaryotic cells and will be limited only by their ability to express active enzymes for their 1,3-propanediol pathway. Suitable host cells include Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Szezocaromyces, Jizosaccharomyces, Peachia, Bacteria such as Kluyveromyces, Candida, Hansenula, Devarios, Mucor, Torulofsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. Preferred host cells for the present invention are Escherichia coli, Escherichia blattae , Klebsiella, Citrobacter , and Aerobacter .
[144] Using the following conventional protocol, microorganisms can be converted to high titer 1,3-propanediol producers.
[145] 1. In a possible host organism, the presence or absence of endogenous dhaT-like activity in the presence of 1,3-propanediol 1-2 M to bring the toxicity or inhibitory level of 3-HPA to a steady state level. Decide;
[146] 2. If the viable host organism has said activity, appropriate mutagenesis is performed to delete or inactivate this activity. Nonfunctional or deleted dhaT-like activity can be demonstrated by detecting the absence of 3-HPA accumulation in the presence of 1,3-propanediol 1-2 M;
[147] 3. If a) glycerol is not a carbon source, express a suitable gene for glycerol production, b) a glycerol dehydratase and a related gene for its associated maintenance system, and c) yqhD.
[148] For certain microorganisms, it is necessary to consider whether to express or inhibit endogenous dhaT-like enzymes under conditions for 1,3-propanediol production. It may also include the presence of glycerol, glucose, or anaerobic conditions.
[149] Vector and Expression Cassettes
[150] The present invention provides a variety of vectors and transformants and expression cassettes suitable for cloning, transforming and expressing G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factors into suitable host cells. Suitable vectors will be vectors compatible with the microorganism employed. Suitable vectors can be from bacteria, viruses (eg, phage derived from bacteriophage T7 or M-13), cosmids, yeasts or plants and the like. Protocols for obtaining and using such vectors are known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual-volumes 1, 2, 3, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989).
[151] Typically, the vector or cassette contains sequences that direct the transcription and translation of the appropriate gene, selectable markers, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors include the 5 'region of the gene, which holds the transcription initiation control region, and the 3' region of the DNA fragment, which controls transcription termination. Most preferably, both control regions are derived from genes homologous to the transformed host cell. The control region need not be derived from a gene that is natural to the particular species selected as production host.
[152] There are a number of initiation control regions, or promoters, useful for driving the expression of G3PDH and G3P phosphatase genes (DAR1 and GPP2, respectively) in a host cell of interest and are known to those skilled in the art. Indeed, any promoter capable of driving such genes is suitable for the present invention, including CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (Saka) Useful for expression in Romanises); AOX1 (useful for expression in pichia); And lac, trp, APL, XPR, T7, tac, and trc (useful for expression in Escherichia coli), and the like.
[153] The termination control region may also be derived from various genes that are natural to the preferred host. In some cases, the termination site may be unnecessary, but it is most preferred that the termination site is included.
[154] For effective expression of the enzymes of the invention, the DNA encoding the enzymes is operably linked through an initiation codon to a selected expression control region and expressed in the form of a suitable mRNA.
[155] Particularly useful in the present invention are the vectors pDT29 and pKP32 designed for use in conjunction with pAH48. Essential elements of pDT29 and pKP32 are derived from the dha regulators isolated from Klebsiella pneumoniae. pDT29 contains the open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, and dhaB3, whose nucleotide sequences are set forth in SEQ ID NO: 1. pKP32 contains the same set of open reading frames as identified for pDT29 from the same source, except that there is no dhaT. pAH48 is a carrier used to introduce the DARI and GPP2 genes into host cells and more specifically includes the DAR1 and GPP2 genes isolated from Saccharomyces cerevisiae.
[156] Transformation and gene expression of suitable hosts for 1,3-propanediol production
[157] Once a suitable cassette is constructed, it is used to transform the appropriate host cell. Introduction of cassettes containing genes encoding G3PDH, G3P phosphatase, dehydratase, and dehydratase reactivation factors into host cells can be achieved by transformation (e.g., using cells that have become permeable using calcium). Perforation) or transfection with recombinant phage virus [Sambrook et al., Supra].
[158] In the present invention, E. coli was transformed using a cassette, as described in detail in the following <General Methods> and <Examples> columns.
[159] Mutant
[160] In the present invention, it is contemplated that in addition to the cells exemplified above, cells with one or multiple mutations, which are specifically designed to enhance the production of 1,3-propanediol, can be used. Typically, these phenotype defects can be prevented by bypassing the carbon feedstock by a non-productive route or by mutating cells that exhibit significant catabolic inhibitory activity. For example, many wild-type cells are susceptible to catabolism inhibition from glucose and by-products in the medium, and mutant strains of such wild-type organisms, which are resistant to glucose inhibition and capable of producing 1,3-propanediol, are particularly useful in the present invention. It is considered useful.
[161] Mutant construction methods are conventional and are known in the art. For example, wild-type cells can be exposed to various agents, such as radiation or chemical mutagenesis, and then the desired phenotype can be screened. In the case of mutation via radiation, ultraviolet (UV) or ionizing radiation can be used. Suitable shortwave UV wavelengths for genetic mutations are in the range of 200 nm to 300 nm, preferably 254 nm. UV radiation of this wavelength mainly changes guanidine and cytosine in the nucleic acid sequence to adenine and thymidine. Because all cells have a DNA repair mechanism that repairs UV-induced mutations, usually, agents such as caffeine and other inhibitors can be added to disrupt the repair process and maximize the number of effective mutations. In addition, mutations by long-wave UV using light in the range from 300 nm to 400 nm are possible, but are typically performed using short-wave UV light unless used in combination with various active agents such as psoralen dyes that interact with DNA. Not as effective as the case
[162] In addition, mutagenesis using chemical agents is effective for mutant production, and commonly used materials include chemicals that affect DNA that is not being replicated, such as HNO ₂ and NH ₂ OH, and acridine dyes. There are agents that affect DNA that is being replicated and are excellent at inducing frameshift mutagenesis. Specific methods of mutant construction using radiation or chemical agents are documented in the art. See, eg, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. Or Deshpande, Mukund V., Appl. Biochem. Biotechnol. 36, 227 (1992).
[163] After mutagenesis, the mutants with the desired phenotype can be selected in a variety of ways. Random screening is the most common method of selecting mutated cells for the desired product or intermediate productivity. Alternatively, selective isolation of the mutants can be performed by propagating a mutated population on a selection medium in which only resistant colonies can proliferate. Mutant selection methods are highly developed and known in the field of industrial microbiology. See, eg, Brock, supra, DeMancilha et al., Food Chem. 14, 313 (1984).
[164] In addition, elimination of unwanted enzyme activity can also be achieved by destroying the gene encoding the enzyme. Such methods are known to those skilled in the art and are exemplified in Examples 4 and 8.
[165] Changes to the 1,3-propanediol Production Route
[166] Representative Enzyme Pathway: 1,3-propanediol production from glucose can be achieved by the following series of steps. This series of steps is representative of numerous routes known to those skilled in the art and is described in FIG. 5. Glucose is converted to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG) via a series of steps by enzymes of the pathway. The DHAP is then reduced after hydrolysis to dihydroxyacetone (DHA) or the DHAP is reduced to glycerol 3-phosphate (G3P) followed by hydrolysis to form glycerol. The hydrolysis step can be catalyzed by any number of intracellular phosphatase, known to be nonspecific to the substrate, or by phosphatase activity introduced into the host by recombination. The reduction step can be catalyzed by NAD ⁺ (or NADP ⁺ ) -binding host enzymes or activity that can be introduced into the host by recombination. It can be noted that the dha regulator can contain glycerol dehydrogenase (EC1.1.1.6) which catalyzes the reversible reaction of Scheme 3 below.
[167] <Scheme 1>
[168] Glycerol → 3-HPA + H ₂ 0
[169] <Scheme 2>
[170] 3-HPA + NADH + H ⁺ → 1,3-propanediol + NAD ⁺
[171] <Scheme 3>
[172] Glycerol + NAD ⁺ → DHA + NADH + H ⁺
[173] As detailed above, the glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxypropionaldehyde (3-HPA). Intermediate 3-HPA produced from glycerol (Scheme 1) is produced by a dehydratase enzyme, which may be encoded by the host or introduced into the host by recombination. The dehydratase may be glycerol dehydratase (EC4.2.1.30), diol dehydratase (EC4.2.1.28) or any other enzyme capable of catalyzing such modifications. Glycerol dehydratase is encoded by dha regulation, while diol dehydratase is not. 1,3-propanediol (Scheme 2) produced from 3-HPA is produced by NAD ⁺ (or NADP ⁺ ) -binding host enzyme or activity that can be introduced into the host by recombination. The final reaction to produce 1,3-propanediol can be catalyzed by 1,3-propanediol dehydronase (EC1.1.1.202) or other alcohol dehydrogenases.
[174] Mutations and transformations affecting carbon channeling
[175] Various mutant microorganisms, including variations in the 1,3-propanediol production pathway, will be useful in the present invention. For example, the introduction of triosphosphate isomerase mutations (tpi-) into the microorganisms of the present invention is an example of using mutants to improve performance by carbon channeling. Triosphosphate isomerase is an enzyme responsible for the conversion of DAHP to 3-phosphoglyceraldehyde and may bypass the main pathway carbon flow from glucose to glycerol and 1,3-propanediol (FIG. 5). . Therefore, the deletion mutation (tpi-) improves the overall metabolic efficiency of the desired pathway than that described in the art. Similarly, mutations that block other pathways to intermediates of the 1,3-propanediol production pathway are also useful in the present invention. For example, by removing glycerol kinase, the action of G3P phosphatase prevents glycerol formed from G3P from converting to G3P, consuming ATP (Figure 5). In addition, removal of glycerol dehydrogenase (eg gldA) results in the conversion of glycerol formed from DHAP to dihydroxyacetone by the action of NADH-dependent glycerol-3-phosphate dehydrogenase (FIG. 5). ) Is prevented. Mutations can be directed against structural genes to impair or improve enzyme activity, or directed against regulatory genes such as promoter regions and ribosomal binding sites, to modulate the expression level of enzyme activity.
[176] Therefore, it is contemplated that certain enzyme activities can be controlled by combining transformations and mutations to enhance 1,3-propanediol production. Thus, it is within the scope of this invention to anticipate modifications of intact cellular catalysts that result in increased production of 1,3-propanediol.
[177] The present invention utilizes a preferred route for producing 1,3-propanediol from sugar substrates, where the carbon flow shifts from glucose to DHAP, G3P, glycerol, 3-HPA, and finally 1,3-propanediol do. The production strains of the present invention can be engineered to incorporate various deletion mutations that prevent the bypass of carbon to non-productive compounds to maximize the metabolic activity of these pathways. As described above, by transformation, glycerol can be bypassed to DHA or G3P via glycerol dehydrogenase or glycerol kinase without conversion to 3-HPA (FIG. 5). Therefore, the production strains of the invention contain deletion mutations in the gldA and glpK genes. Similarly, DHAP can be bypassed to 3-PG by triosphosphate isomerase so that the producing microorganism of the present invention also contains a deletion mutation in this gene. The method also incorporates a dehydratase enzyme that converts glycerol to 3-HPA, which functions with the reactivation factors encoded by orfX and orfZ of the dha regulator (FIG. 5). The conversion of 3-HPA to 1,3-propanediol is typically carried out via 1,3-propanediol oxidoreductase, but the process of the present invention provides a titer for the production of 1,3-propanediol, the final product, and Higher yields use nonspecific catalytic activity (FIG. 5). In this method, titers to 1,3-propanediol are achieved at 10 g / L or more, with a titer of 200 g / L expected.
[178] Alternatively, an improved method for the production of 1,3-propanediol uses glycerol or dihydroxyacetone as substrates, with glycerol → 3-HPA → 1,3-propanediol whose route only includes the last three substrates. Can be. In this method, oxidoreductase is removed again for nonspecific catalytic activity (predicted to be an alcohol dehydrogenase), but adding glycerol to the culture eliminates the deletion mutation. In this method, titers to 1,3-propanediol are achieved above 71 g / L, with a titer of 200 g / L expected.
[179] Similarly, the scope of the present invention includes providing mutants of wild type microorganisms modified or deleted by mutating dhaT activity for improved 1,3-propanediol producer construction. For example, microorganisms that naturally contain all elements of the dha regulator can be engineered to inactivate the dhaT gene encoding 1,3-propanediol oxidoreductase activity. The microorganisms will be expected to have higher yields and titers for 1,3-propanediol production mediated by endogenous catalytic activity (predicted by alcohol dehydrogenase). Examples of the microorganism include, but are not limited to, Klebsiella spp., Citrobacter spp., And Clostridium spp.
[180] Badges and Carbon Substrates
[181] The fermentation medium in the present invention should contain a suitable carbon substrate. Suitable substrates include monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof, and cheese whey permeate, cornsteep liquid, sugar beet molasses and malt ( crude mixtures from renewable feedstocks such as barley malt), and the like. The carbon substrate may also be a 1-carbon substrate that has been demonstrated for metabolic conversion to key biochemical intermediates such as carbon dioxide or methanol. Methyl nutritional yeast [K. Yamada et al., Agric. Biol. Chem. 53 (2), 541-543 (1989) and bacteria [Hunter et. al., Biochemistry 24, 4148-4155 (1985), reported the production of glycerol from a 1-carbon source (eg, methanol, formaldehyde or formate). The microorganisms can assimilate 1-carbon compounds from methane to formate in an oxidized state to produce glycerol. The carbon assimilation pathway can be via ribulose monophosphate, serine or xylose-monophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag: New York (1986)). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5-phosphate to form hexasaccharides, which are fructose, and the formation of glyceraldehyde-3-phosphate, which is in fact a trisaccharide product. Similarly, the serine pathway assimilates the 1-carbon compound through the methylenetetrahydrofolate to that pathway.
[182] In addition to carbon and monosaccharide carbon substrates, methyltrophic microorganisms are also known to utilize numerous other carbon-containing compounds such as methylamine, glucoseamine and various amino acids for metabolic activity. For example, methyltrophic yeast is known to form trehalose or glycerol using carbon from methylamines [Bellion et al., Microb. Growth C1 Compd, Int. Symp.], 7th (1993), 415-32. Editor (s); Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK. Similarly, various Candida species will metabolize alanine or oleic acid [Sulter et al., Arch. Microbiol. 153 (5), 485-489 (1990)]. Therefore, it is contemplated that the carbon source used in the present invention may encompass a wide variety of carbon containing substrates and will be limited only by the microorganisms or methods selected.
[183] Although all of the above-mentioned carbon substrates and mixtures thereof (simultaneous feeds) are considered suitable for the present invention, preferred carbon substrates are glucose, fructose, sucrose, or methanol (this process produces endogenous glycerol), and Glycerol or dihydroxyacetone, which is expected to produce glycerol or dihydroxyacetone.
[184] The fermentation medium should contain not only a suitable carbon source, but also suitable minerals, salts, cofactors, buffers and other components suitable for the promotion of the enzymatic pathways required for the production of 1,3-propanediol and for propagation of the culture, as known to those skilled in the art. . Of particular interest are Co (II) salts and / or vitamin B ₁₂ or precursors thereof.
[185] Adenosyl-cobalamin (coenzyme B ₁₂ ) is an essential cofactor for dehydratase activity. Synthesis of coenzyme B ₁₂ is found in prokaryotes, for example, some prokaryotes such as Escherichia blata, Klebsiella spp., Citrobacter spp., And Clostridium spp. ( de novo ), but some may perform partial reactions. For example, E. coli cannot produce a corinated ring structure, but can catalyze the conversion of corbinamide to corinoids and introduce 5'-deoxyadenosyl groups. Therefore, it is known in the art that it is necessary to provide coenzyme B ₁₂ precursors such as vitamin B ₁₂ during E. coli fermentation.
[186] The addition of vitamin B ₁₂ to E. coli fermentation can be added continuously at a constant rate, stepwise depending on the resulting cell mass, or added all at once or multiple times. The preferred ratio of vitamin B ₁₂ feed (mg) to cell mass (OD ₅₅₀ ) is 0.06 to 0.60. The most preferred ratio of vitamin B ₁₂ feed (mg) to cell mass (OD ₅₅₀ ) is 0.12 to 0.48.
[187] Even if vitamin B ₁₂ is added to the transformed E. coli of the present invention, it is contemplated that other microorganisms capable of de novo biosynthesis of B ₁₂ are also suitable production cells, and that B ₁₂ need not be added to these microorganisms.
[188] Culture conditions :
[189] Typically, cells are grown in suitable medium at 35 ° C. Preferred growth media of the present invention are conventionally prepared media, such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or yeast medium (YM) broth and the like. Other defined or synthetic growth media may also be used and suitable media for the growth of certain microorganisms are known to those skilled in the art of microbiology or fermentation science. In addition, agents known to directly or indirectly regulate catabolic metabolism inhibition, such as cyclic adenosine 2 ′: 3′-monophosphate, may also be incorporated into the reaction medium. Similarly, agents known to modulate enzymatic activity (eg, methyl viologen) can be used as an alternative to or in conjunction with genetic manipulation to enhance 1,3-propanediol production. .
[190] Suitable pH ranges for fermentation are between pH 5.0 and pH 9.0, and preferred ranges of initial conditions are pH 6.0 to pH 8.0.
[191] It can be reacted under aerobic or anaerobic conditions, and is preferably anaerobic or microaerobic conditions.
[192] Fed-Batch fermentation can be carried out using a carbon feed such as glucose in limited or excessive amounts.
[193] Batch and Continuous Fermentation :
[194] The method of the present invention uses a batch fermentation method. Classical batch fermentation is a closed system which does not artificially alter the medium composition established at the start of fermentation during fermentation. Therefore, at the beginning of fermentation, the medium is inoculated with the desired microorganism (s) and fermented, and nothing is added to this system. Typically, however, a "batch" fermentation is a batch for the addition of a carbon source and often requires adjustment of factors such as pH and oxygen concentration. In a batch system, the metabolite and biomass composition in the system changes continuously until the fermentation is stopped. In batch cultures, cell proliferation proceeds from a static lag phase to a high log phase, eventually resulting in a stationary phase in which the rate of proliferation is reduced or stopped. If untreated, the stationary cells will virtually die. Typically, logarithmic cells produce most of the final product or intermediate.
[195] A variation of the standard batch system is the Fed-Batch system. Ped-batch fermentation methods are also suitable for the present invention and include the manner of typical batch systems, except that the amount of substrate added increases as the fermentation proceeds. If metabolic inhibition tends to inhibit cell metabolism and it is desirable to limit the amount of substrate in the medium, a fed-batch system is useful. Since it is difficult to measure the actual substrate concentration in the ped-batch system, it is estimated based on changes in measurable factors such as pH, dissolved oxygen and partial pressure of waste gases such as CO ₂ . Batch and fed-batch fermentations are conventional and known in the art, examples of which can be found in Brock, supra.
[196] Even if the present invention is carried out batchwise, it is contemplated that this method could be adapted to a continuous fermentation method. Continuous fermentation is an open system that continuously adds a defined fermentation medium to the bioreactor while simultaneously removing the same amount of condition medium for processing. Typically, continuous fermentation maintains the culture at a constant high density at which cells are primarily in logarithmic proliferation.
[197] Continuous fermentation can adjust one or any number of factors that affect cell proliferation or final product concentration. For example, one method can adjust all other parameters while limiting nutrient or nitrogen levels such as carbon sources to fixed values. In other systems, the cell concentration, as measured by medium turbidity, can be continuously varied with many factors affecting proliferation while maintaining a constant. Since continuous systems seek to maintain steady state growth conditions, cell loss due to media recovery should be balanced with the rate of cell proliferation during fermentation. Methods for adjusting nutrients and growth factors for continuous fermentation methods and techniques for maximizing product formation rates are known in the industrial microbial art and various methods are described in detail in Brock, supra.
[198] The present invention can be carried out using batch, fed-batch or continuous methods, and it is contemplated that any known mode of expression is suitable. It is also contemplated that cells can be immobilized on a substrate as intact cellular catalysts under fermentation conditions for 1,3-propanediol production.
[199] Identification and purification of 1,3-propanediol :
[200] Methods of purifying 1,3-propanediol from fermentation media are known in the art. For example, the reaction mixture can be extracted, distilled, and column chromatographed with an organic solvent to obtain propanediol from the cell medium (US Pat. No. 5,356,812). A particularly preferred organic solvent for this process is cyclohexane [US Pat. No. 5,008,473].
[201] The medium can be analyzed by high pressure liquid chromatography (HPLC) to directly identify 1,3-propanediol. A preferred method for the present invention is to analyze the fermentation medium in an isocratic fashion on an analytical ion exchange column using 0.01 N sulfuric acid as the mobile phase.
[202] Example
[203] The usual way
[204] Phosphorylation, ligation and transformation methods are known in the art. Techniques suitable for use in the examples below can be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).
[205] Materials and methods suitable for the maintenance and propagation of bacterial cultures are known in the art. Techniques suitable for use in the following examples are described in Manual of Methods for General Bacteriology (Phillipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds). , American Society for Microbiology, Washington, DC (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. Unless otherwise noted, all reagents and materials used for the growth and maintenance of bacterial cells are from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO / BRL (Gaytersberg, MD), or Sigma Chemical Company (St. Louis, MO).
[206] The abbreviation means: "hour (h)" means hour (s), "minute" means minute (min), "second" means second (sec), "Day" means days, "mL" means milliliters (mL), "L" means liters (L), 50 amps is 50 μg / mL and LB-50 amps are Luria Bertani Broth containing 50 μg / mL Ampicillin.
[207] The following abbreviations in the table were used to mean the following: "Con." Means conversion, "Sel." Means selectivity based on carbon, and "nd" was not detected.
[208] The strains and vectors used in the following examples are listed in the following chart:
[209]
[210] Enzyme analysis
[211] Dehydratase Enzyme Assay :
[212] Dehydratase activity in cell-free extracts was measured using glycerol or 1,2-propanediol as substrate. Typically, cell-free extracts were prepared by disrupting cells using a french press, followed by centrifugation of intracellular debris. This assay based on the reaction of aldehydes with methylbenzo-2-thiazolone hydrazone is described in Forage and Foster, Biochim. Biophys. Acta 569, 249 (1979).
[213] Honda et al., J. Bacteriol. 143, 1458 (1980), discloses assays for measuring the reactivation rate of dehydratase. Dehydratase activity in toluenized intact cells was measured using glycerol or 1,2-propanediol as substrate and with and without ATP. The reactivation rate was measured as the product formation rate with and without ATP. Product formation (3-HPA or propionaldehyde, respectively, when glycerol or 1,2-propanediol is used as the substrate) can be measured directly using HPLC or indirectly using methylbenzo-2-thiazolone hydrazone reagent did. Alternatively, NADH-linked alcohol dehydrogenase was used to couple the conversion of the aldehyde to its corresponding alcohol, and the NADH disappearance was monitored to determine product formation.
[214] 1,3-propanediol oxidoreductase assay :
[215] Johnson and Lin, J. Bacteriol. 169, 2050 (1987), using 1,3-propanediol and NAD ⁺ as substrates for 1,3-propanediol oxidoreductases on cell-free extracts in solution or slab gel ( Sometimes referred to as 1,3-propanediol dehydrogenase). Alternatively, conversion of 3-HPA and NADH to 1,3-propanediol and NAD ⁺ was determined by the disappearance of NADH. Slab gel assay has the strong advantage of separating the activity of 1,3-propanediol oxidoreductase (dhaT) from nonspecific alcohol dehydrogenase activity by size separation method. The natural molecular weights of 1,3-propanediol oxidoreductase (dhaT) from Citrobacter Proindy, Klebsiella pneumoniae, and Clostridium pasteerianum are typically in the range of 330,000 to 440,000 Da. Lactobacillus brevis and Lactobacillus bukneri contain 1,3-propanediol oxidoreductase-related dehydratase with similar characteristics to the known 1,3-propanediol oxidoreductase (dhaT).
[216] Glycerol 3-phosphate dehydrogenase Activity Assay :
[217] Bell et al., J. Biol. Chem. 250, 7153 (1975), was used to modify the method as follows. This method comprises a sample of cell-free extract in qubit containing 0.2 mM NADH, 2.0 mM dihydroxyacetone phosphate (DHAP), and enzymes in 0.1 M Tris / HCl (pH 7.5) buffer and 5 mM DTT (total volume 1.0 mL). Incubating at 30 ° C. First, the background rates of the enzyme and NADH reactions were measured for at least 3 minutes at 340 nm. Then, a second substrate, DHAP, was added and the absorbance change over time was continuously monitored for at least 3 minutes. G3PDH activity was defined as full speed minus background speed.
[218] Glycerol-3-phosphatase activity assay :
[219] Enzyme activity assays were performed by incubating the extract with an organic phosphate substrate in bis-tris or MES and magnesium buffer, pH 6.5. The substrate used was 1-α-glycerol phosphate, or d, 1-α-glycerol phosphate. Final concentrations of reagents used in this assay were as follows: buffer (20 mM Bis-Tris or 50 mM MES), MgCl ₂ (10 mM), and substrate (20 mM). If the total amount of protein in the sample was low and there was no visible precipitate due to acid quenching, the sample was conveniently analyzed in qubits. The method includes incubating the enzyme sample in qubits containing 20 mM substrate (50 μl, 200 mM), 50 mM MES, 10 mM MgCl ₂ (pH 6.5) buffer. The final phosphatase assay volume was 0.5 mL. Enzyme-containing samples were added to the reaction mixture; After the contents in the qubits were mixed, the qubits were placed in a circulating water bath at temperature = 37 ° C. for 5 to 120 minutes (this time depends on the phosphatase activity in the enzyme sample (range of 2 to 0.02 U / mL)). The enzyme reaction was stopped by quenching by addition of acid molybdate reagent (0.4 mL). After addition of the Fiske SubbaRow reagent (0.1 mL) and distilled water (1.5 mL), the solution was mixed and developed. After 10 minutes complete color development appeared and the absorbance of the sample was measured at 660 nm using a Cary 219 UV / BIS spectrophotometer. The stock inorganic phosphate solution (0.65 mM) was used to compare the amount of inorganic phosphate released with a standard curve made from six standards made with final inorganic phosphate concentrations ranging from 0.026 to 0.130 μmol / mL.
[220] Glycerol Kinase Activity Assay :
[221] At 25 ° C., an appropriate amount of enzyme, typically cell-free crude extract, was added to 40 mM ATP, 20 mM MgSO ₄ , 21 mM homogeneous ¹³ C-labeled glycerol (99%, manufactured by Cambridge Isotope Laboratories), and To the reaction mixture containing 0.1 M Tris-HCl (pH 9) was added for 75 minutes. Conversion of glycerol to glycerol 3-phosphate was detected by ¹³ C-NMR (125 MHz): glycerol (63.11 ppm, δ, J = 41 Hz and 72.66 ppm, t, J = 41 Hz); Glycerol 3-phosphate (62.93 ppm, δ, J = 41 Hz; 65.31 ppm, br d, J = 43 Hz; and 72.66 ppm, dt, J = 6, 41 Hz).
[222] NADH-linked glycerol dehydrogenase assay :
[223] After protein isolation by unmodified polyacrylamide gel electrophoresis, NADH-linked glycerol dehydrogenase activity (gldA) in cell-free extracts from E. coli strains was measured. Conversion of glycerol + NAD ⁺ to dihydroxyacetone + NADH using phenazine methosulfate (PMS) as a mediator was carried out using 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetra Coupling of Zolium Bromide (MTT) with the conversion to dark color formazan [Tang et al., J. Bacteriol. 140, 182 (1997).
[224] Electrophoresis was performed in duplicate using a standard method using natural gels (8-16% TG, 1.5 mm, gel with 15 lanes, Novex, San Diego, Calif.). Using 50 mM Tris or potassium carbonate buffer (pH 9), the gel was washed three times for 10 minutes to remove residual glycerol. Under conditions with and without glycerol (final concentration: approximately 0.16 M), the duplicate gels were treated with 50 mM tris or potassium carbonate (pH 9), 60 mg ammonium sulfate, 75 mg NAD ⁺ , 1.5 mg MTT, and 0.5 mg It was developed in 15 mL of assay solution containing PMS.
[225] In addition, after polyacrylamide gel electrophoresis, NADH-binding glycerol dehydrogena in E. coli strain (gldA) was reacted with a polyclonal antibody directed against purified Klebsiella pneumoniae glycerol dehydrogenase (dhaD). The presence of the first activity was determined.
[226] Isolation and Identification of 1,3-propanediol :
[227] The conversion of glycerol to 1,3-propanediol was monitored by HPLC. Analyzes were performed using standard techniques and materials known to those skilled in the art of chromatography. One suitable method used a Waters Maxima 820 HPLC system using UV (210 nm) and RI detection. The temperature of the Shodex SH-1011 column (8 mm × 300 mm, Waters, Milford, Mass.), Equipped with a Shodex SH-1011P precolumn (6 mm × 50 mm), is 50 ° C. The sample was injected into the sample, 0.01 NH ₂ SO ₄ was used as the mobile phase, and the flow rate was 0.5 mL / min. If quantitative analysis is desired, the samples were prepared using known amounts of trimethylacetic acid as an external standard. Typically, the residence times of glucose (RI detection), glycerol, 1,3-propanediol (RI detection), and trimethylacetic acid (UV and RI detection) were 15.27 minutes, 20.67 minutes, 26.08 minutes, and 35.03 minutes, respectively.
[228] Production of 1,3-propanediol was demonstrated using GC / MS. Analyzes were performed using standard techniques and materials available to those skilled in the GC / MS art. One suitable method is the Hewlett Packard 5890 Series II gas chromatography coupled with Hewlett Packard 5971 Series mass selection detector (EI) and HP-INNOWax columns (30 m long, 0.25 mm inner diameter, 0.25 micron thick). I'm using photography. The residence time and mass spectrum of the resulting 1,3-propanediol were compared with the values of certified 1,3-propanediol (m / e: 57, 58).
[229] Alternative methods for GC / MS included derivatization of the sample. 30 μl of concentrated perchloric acid (70% v / v) was added to 1.0 mL of the sample (eg, culture supernatant). After mixing, the sample was frozen and lyophilized. To the lyophilized material was added a 1: 1 mixture of bis (trimethylsilyl) trifluoroacetamide: pyridine (300 μl), mixed vigorously and left at 65 ° C. for 1 hour. Samples were centrifuged to remove insoluble material to clear the samples. A liquid partitioned into two phases was produced, of which the supernatant was analyzed. This sample was chromatographed on a DB-5 column (48 m, inside diameter 0.25 mm, membrane thickness 0.25 μm; J & W Scientific) and obtained from the 1,3-propanediol derivative obtained from the culture supernatant. Retention time and mass spectra were compared with values obtained from certified standards. The mass spectrum of TMS-derived 1,3-propanediol contained characteristic ions of 205, 177, 130 and 115 AMU.
[230] Cell Lysis :
[231] Cell lysis was assessed by measuring extracellular soluble protein concentration in fermentation broth. Fermentation samples were centrifuged in a desktop centrifuge (typically running for 3-5 minutes at 12,000 rpm in a Model 5415C micro centrifuge from Eppendorf) to separate cells. The protein concentration of the resulting supernatants was analyzed using the Bradford method using commercial reagents (Bio-Rad protein analysis, Bio-Rad, Hercules, Calif.).
[232] Viability :
[233] Cell viability was determined by appropriate dilution of cells obtained from the fermentor and plating on non-selective LB agar plates. Cell viability between fermentation experiments was compared using the ratio of viable cells per mL of fermentation broth by OD ₅₅₀ (AU).
[234] Example 1
[235] Cloning and Transformation of Escherichia Coli Host Cells Using Cosmid DNA for Expression of 1,3-propanediol
[236] Badge :
[237] Synthetic S12 medium was used for screening bacterial transformants for 1,3-propanediol production capacity. S12 medium contains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer (pH 7.0), 2 mM MgCl ₂ , 0.7 mM CaCl ₂ , 50 μM MnCl ₂ , 1 μM FeCl ₃ , 1 μM ZnCl, 1.7 μM CuCO ₄ , 2.5 μΜ CoCl ₂ , 2.4 μΜ Na ₂ MoO ₄ , and 2 μΜ thiamine hydrochloride.
[238] Medium A used for propagation and fermentation consists of: 10 mM ammonium sulfate; 50 mM MOPS / KOH buffer, pH 7.5, 5 mM potassium phosphate buffer, pH 7.5, 2 mM MgCl ₂ , 0.7 mM CaCl ₂ , 50 μM MnCl ₂ , 1 μM FeCl ₃ , 1 μM ZnCl, 1.72 μM CuSO ₄ , 2.53 μM CoCl ₂ , 2.4 μM Na ₂ MoO ₄ , 2 μM Thiamine Hydrochloride, 0.01% Yeast Extract, 0.01% Casamino Acid, 0.8 μg / mL Vitamin B ₁₂ , and 50 μg / mL Ampicillin. If necessary, medium A may be supplemented with 0.2% glycerol or 0.2% glycerol and 0.2% D-glucose.
[239] Cells :
[240] Klebsiella pneumoniae ECL2106 also known as Klebsiella aerogenes or Aerobacter aerogenes in the literature [Ruch et al, J. Bacteriol. 124, 348 (1975) was obtained from ECCLin (Harvard Medical School, Massachusetts, USA) and maintained as a laboratory culture.
[241] Klebsiella pneumoniae ATCC25955 was purchased from the American Type Culture Collection (Manassas, VA, USA).
[242] E. coli DH5α was purchased from Gibco / BRL and transformed with cosmid DNA containing genes encoding glycerol or diol dehydratase enzymes isolated from Klebsiella pneumoniae ATCC 25955. Cosmids containing glycerol dehydratase were referred to as pKP1 and pKP2, and cosmids containing diol dehydratase enzyme were referred to as pKP4. Transformed DH5α cells were referred to as DH5α-pKP1, DH5α-pKP2, and DH5α-pKP4.
[243] E. coli ECL707 [Sprenger et al., J. Gen. Microbiol. 135,1255 (1989)] was obtained from ECCLin (Harvard Medical School, Massachusetts, USA, and similarly transformed using cosmid DNA from Klebsiella pneumoniae. The transformants containing the hydratase gene were referred to as ECL707-pKP1 and ECL707-pKP2 and the transformants containing the diol dehydratase gene were referred to as ECL707-pKP4.
[244] E. coli AA200 containing a mutation in the tpi gene [Anderson et al., J. Gen. Microbiol. 62, 329 (1970)] were purchased from the E. coli Genetic Stock Center at Yale University (New Haven, Connecticut, USA), using the Klebsiella cosmid DNA. The conversion resulted in recombinant microorganisms AA200-pKP1 and AA200-pKP2 containing the glycerol dehydratase gene and recombinant microorganism AA200-pKP4 containing the diol dehydratase gene,
[245] DH5α :
[246] Six transformation plates containing approximately 1,000 E. coli XL1-Blue MR colonies transfected with Klebsiella pneumoniae DNA were washed with 5 mL LB medium and centrifuged. The bacteria were pelleted and resuspended in 5 mL LB medium + glycerol. Aliquots (50 μl) were inoculated into a 15 mL tube containing S12 synthetic medium with 0.2% glycerol + vitamin B ₁₂ 400 ng / mL + 0.001% yeast extract + 50 amp. The tube was filled to the end with the medium, wrapped in parafilm and incubated at 30 ° C. After 48 hours, some suspension was observed. As described above, when aliquots were analyzed for the product at 78 and 132 hours, these aliquots were positive for 1,3-propanediol, and at 132 hours the amount of 1,3-propanediol was Increased.
[247] As a result of the tests, single colonies were isolated by serial dilutions of bacteria that showed positive for 1,3-propanediol production and plating on LB-50 amp plates. 48 single colonies were isolated and reviewed for 1,3-propanediol production. Cosmid DNA was isolated from six independent clones and used to transform E. coli strain DH5α. This transformant was again evaluated for 1,3-propanediol production. Two transformants were further characterized and referred to as DH5α-pKP1 and DH5α-pKP2.
[248] The 12.1 kb EcoRI-SalI fragment of pKP1 was subcloned into pIBI31 (IBI Biosystem, New Haven, Connecticut) and sequenced to pHK28-26 (SEQ ID NO: 1). The sequencing revealed the loci of the genes required for the related open reading frame and regulation of the dha operon encoding the open reading frame glycerol dehydratase.
[249] In relation to SEQ ID NO: 1, an open reading frame fragment of dhaK1 encoding a dihydroxyacetone kinase was found at bases 1-399 (complement); An open reading frame dhaD encoding glycerol dehydrogenase was found at bases 1010-2107; An open reading frame dhaR encoding the repressor was found at bases 2209-4134; An open reading frame orfW encoding an unknown function protein was found at bases 4112-4642 (complement); An open reading frame orfX encoding dehydratase reactivation protein was found at bases 4643-4996 (complement); An open reading frame dhaT encoding 1,3-propanediol oxidoreductase was found at bases 5017-6180 (complement); An open reading frame orfY encoding an unknown function protein was found at bases 6202-6630 (complement); the open reading frame dhaB1 encoding α subunit glycerol dehydratase was found at bases 7044-8711; an open reading frame dhaB2 encoding β subunit glycerol dehydratase was found at bases 8724-9308; γ subunit glycerol dehydratase open reading frame dhaB3 was found at bases 9311-9736; An open reading frame dhaBX encoding dehydratase reactivation protein was found at bases 9749-11572; A glpF open reading frame fragment encoding a glycerol uptake promoter protein was found at bases 11626-12145.
[250] A single colony of Escherichia coli XL1-Blue MR transfected with cosmid DNA packaged from Klebsiella pneumoniae was treated with 200 μl of S15 medium (ammonium sulfate, 10 mM; potassium phosphate buffer, pH 7.0, 1 mM; MOPS / KOH Buffer, pH 7.0, 50 mM; MgCl ₂ , 2 mM; CaCl ₂ , 0.7 mM; MnCl ₂ , 50 nM; FeCl ₃ , 1 μM; ZnCl, 1 M; CuSO ₄ , 1.72 μM; CoCl ₂ , 2.53 μM; Na ₂ MoO ₄ , 2.42 μM; and thiamine hydrochloride, 2 μM) + 0.2% glycerol + 400 ng / mL Vitamin B ₁₂ + 0.001% yeast extract + 50 μg / mL ampicillin were also inoculated. In addition to the microtiter wells, inoculated into master plates containing LB-50 amp. After 96 hours, 100 μl was taken and centrifuged in Rainin microfuge containing 0.2 micron nylon membrane filter. The filtrate was analyzed by HPLC leaving bacteria. Approximately 240 colonies were identified after screening positive clones for which 1,3-propanediol production was demonstrated. Three positive clones were identified, two of which grew in LB-50 amp and one clone did not. Single colonies isolated from one of two positive clones cultured on LB-50 amp and demonstrated production of 1,3-propanediol were termed pKP4. Cosmid DNA was isolated from the E. coli strain containing pKP4 and transformed into E. coli strain DH5α. Independent transformants called DH5α-pKP4 were identified for 1,3-propanediol production.
[251] ECL707:
[252] E. coli strain ECL707 was transformed with cosmid Klebsiella pneumoniae DNA and supercos vector alone corresponding to pKP1, pKP2, pKP4, which are referred to as ECL707-pKP1, ECL707-pKP2, ECL707-pKP4 and ECL707-sc, respectively. did. ECL707 has glpK , gld , and ptsD encoding enzymes II for dihydroxyacetones of ATP-dependent glycerol kinase, NAD ⁺ -linked glycerol dehydrogenase and phosphoenolpyruvate dependent phosphotransferases, respectively Is defective.
[253] Twenty single colonies transformed with each cosmid and five colonies (negative controls) isolated from LB-50amp plates were transferred to master LB-50 amp plates. The ability to convert glycerol to 1,3-propanediol was tested to see if they had dehydratase activity. The transformants were transferred to a microtiter plate containing 200 μl of medium A supplemented with 0.2% glycerol or 0.2% glycerol + 0.2% D-glucose with sterile toothpicks. After 48 hours of incubation at 30 ° C., the contents of the microtiter plate wells were filtered through a 0.45 micron nylon filter and chromatographed by HPLC. The results of this test are shown in Table 2.
[254] Conversion of glycerol to 1,3-propanediol by transformed ECL707 Transformant Glycerol * Glycerol + Glucose * ECL707-pKP1 19/20 19/20 ECL707-pKP2 18/20 20/20 ECL707-pKP4 0/20 20/20 ECL707-sc 0/5 0/5 Number of positive isolates / number of tested isolates
[255] AA200:
[256] E. coli strain AA200 was transformed with cosmid Klebsiella pneumoniae DNA and supercos vector alone corresponding to pKP1, pKP2, pKP4, and these were respectively converted to AA200-pKP1, AA200-pKP2, AA200-pKP4 and AA200-sc, respectively. Referred to. Strain AA200 is defective in triosephosphate isomerase ( trp −).
[257] Twenty single colonies transformed with each cosmid and five colonies transformed with the empty vector were isolated and tested for their ability to convert glycerol to 1,3-propanediol as described above for E. coli strain ECL707. did. The test results are shown in Table 3.
[258] Conversion of glycerol to 1,3-propanediol by transformed AA200 Transformant Glycerol * Glycerol + Glucose * AA200-pKP1 17/20 17/20 AA200-pKP2 17/20 17/20 AA200-pKP4 2/20 16/20 AA200-sc 0/5 0/5 Number of positive isolates / number of tested isolates
[259] Example 2
[260] Glycerol Kinase Mutant Manipulation of Escherichia Coli FM5 to Produce Glycerol from Glucose
[261] Integrated Plasmid Construction for Glycerol Kinase Gene Replacement of Escherichia Coli FM5 :
[262] E. coli FM5 (ATCC 53911) genomic DNA was prepared using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn.). 1.0 kb DNA fragments containing a portion of glpF and a glycerol kinase (glpK) gene were purified from FM5 genomic DNA using primer sequences 2 and 3 [Mullis and Faloona, Methods Enzymol. 155, 335 (1987). 1.1 kb DNA fragments containing a portion of glpK and glpX gene were amplified by PCR from FM5 genomic DNA using primer sequences 4 and 5. MunI site was included in primer sequence 4. The 5 'end of primer sequence 4 was complementary in the opposite direction to primer sequence 3, so that subsequent overlap extension PCR could be performed. 2.1 kb by PCR using the two PCR fragments and primer sequences 2 and 5 as template, using gene splicing by the overlap extension technique [Horton et al., BioTechniques 8, 528-535 (1990)] A fragment was obtained. The fragment showed a deletion of 0.8 kb from the central region of the 1.5 kb glpK gene. In total, the fragment had flanking regions of 1.0 kb and 1.1 kb on one side of the MunI cloning site (in the glpt portion), allowing homologous recombination to replace the chromosomal gene.
[263] The 2.1 kb PCR fragment was blunt-ended (using a mung bean nuclease) and a Zero Blunt PCR Cloning Kit (Invitrogen, San Diego, Calif.) Cloning into the pCR-Blunt vector yielded a 5.6 kb plasmid pRN100 containing kanamycin and zeocin resistance genes. MunI-cleaved using a 1.2 kb HincII fragment of pLoxCat1 (unpublished results) containing a chloramphenicol-resistant gene flanking the bacteriophage P1 loxP site (Snaith et al., Gene 166, 173 (1995)) Blunt-ended) was ligated into plasmid pRN100 and inserted between glpK fragments in plasmid pRN100 to give a 6.9 kb plasmid pRN101-1. The 376 bp fragment containing the R6K origin was subjected to PCR using primer sequences 6 and 7 [Miller and Mekalanos, J. Bacteriol. 170, 2575-2583 (1988)], amplified from vector pGP704, blunt ended, and ligated to 5.3 kb Asp7l8-AatII fragment (blunt ended) of pRN101-1 to kanamycin and chloramphenicol resistance genes. A 5.7 kb plasmid pRN102-1 containing was obtained. pRN102-1 was obtained by replacing the ColE1 starting region in pRN101-1 with the R6K starting point, and most of the zeocin resistance genes were deleted. Hosts for pRN102-1 replication include Escherichia coli SY327 [Miller and Mekalanos, J. Bacteriol. 170, 2575-2583 (1988).
[264] Manipulation of the glycerol kinase mutant RJF10m using chloramphenicol resistant gene inserts :
[265] E. coli FM5 was electrotransformed into a non-replicating integrated plasmid pRN 102-1 and M9 minimal medium containing 1 mM glycerol in transformants that are chloramphenicol (12.5 g / mL) resistant and kanamycin (30 μg / mL) sensitive Transformants without glucose in the phase were screened. Southern analysis with complete glpK gene [Southern, J. Mol. Biol. 98, 503-517 (1975), said EcoRI fragment of the mutant, RJF10m genomic DNA, predicted two 7.9 kb and 2.0 kb predicted due to the presence of additional EcoRI sites in the chloramphenicol resistance gene. The band was observed, indicating that it is a double-crossover integration (glpK gene substituent). Wild-type control produced a single predicted 9.4 kb band. ¹³ C NMR analysis of mutant RJF10m has proven that it is possible to convert the ¹³ C- labeled glycerol and ATP to glycerol-3-phosphate. The glpK mutants were further analyzed by genomic PCR using the primer combinations of SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and SEQ ID NO: 8 and SEQ ID NO: 11 to predict the predicted 2.3 kb, 2.4 kb, and 4.0 kb PCR fragments. Each generated. Wild-type controls generated a predicted 3.5 kb band using primers SEQ ID NO: 8 and SEQ ID NO: 11. The glpK mutant RJF10m was electrotransformed into plasmid pAH48 to produce glycerol from glucose. The glpK mutant Escherichia coli RJF10m was deposited with ATCC under the Budapest Treaty on November 24, 1997.
[266] Glycerol Kinase Mutant RJF10 Manipulation Using a Removed Chloramphenicol Resistant Gene Insert :
[267] After overnight incubation at 37 ° C. on YENB medium (0.75% yeast extract, 0.8% nutrient broth), E. coli RJFlOm in an aqueous suspension was subjected to bacteriophage P1 Cre under the control of the IPTG inducible lacUV5 promoter, temperature-sensitive pSC101 replicon and ampicillin resistance gene. Electrotransformation was done with plasmid pJW168 (unpublished result) containing recombinase. With further incubation at 30 ° C. in SOC medium, transformants were selected at 30 ° C. (permissible temperature for pJW168 replication) on LB agar medium supplemented with carbenicillin (50 μg / mL) and IPTG (1 mM). Collected colonies were transferred on fresh LB agar medium supplemented with carbenicillin and IPTG for 2 consecutive nights at 30 ° C. to incubate overnight to generate chromosome chloramphenicol resistance genes by Cre recombinase [Hoess and Abremski, J. Mol. Biol. 181, 351-362 (1985), cleavage of the chromosome chloramphenicol resistance gene by recombination at the loxP site. The resulting colonies were replicated on LB agar medium supplemented with carbenicillin and IPTG and LB agar medium supplemented with chloramphenicol (12.5 μg / mL) to show carbenicillin resistance and marker gene removal. Colonies that were chloramphenicol sensitive were identified. One colony was incubated overnight at 30 ° C. and used to inoculate 10 mL of LB medium. Cultures were incubated at 30 ° C. at OD (600 nm) to 0.6 AU and cultures were incubated overnight at 37 ° C. Plated in pre-warmed LB agar medium at various dilutions and plates were incubated overnight at 42 ° C. (unacceptable temperature of pJW168 replication). The resulting colonies were replicated in LB agar medium supplemented with LB agar medium and carbenicillin (75 μg / mL) to identify carbenicillin sensitive colonies indicating loss of plasmid pJW168. One glpK mutant, RJF 10, was further analyzed by genomic PCR using primer sequences 8 and 11 to produce a predicted 3.0 kb band indicating that the marker gene was cleaved off. The mutant RJF10's inability to utilize glycerol has proven to be unable to grow in M9 minimal medium containing 1 mM glycerol. The glpK mutant RJF10 was electrotransformed into plasmid pAH48 to produce glycerol from glucose.
[268] Example 3
[269] Construction of Escherichia Coli Strains with KldA Gene Knocked Out
[270] The gldA gene of Escherichia coli was isolated by PCR using primers SEQ ID NO: 12 and SEQ ID NO: 13 containing terminal SphI and XbaI sites, respectively, [KB Mullis and FA Faloona, Meth. Enzymol. 155, 335-350 (1987)], cloning between the SphI and XbaI sites in pUC18 [T. Maniatis (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor, NY] to produce pKP8. pKP8 was cleaved at the only SalI and NcoI sites in the gldA gene and the ends were treated with Klenow and ligated again to delete 109 bp in the middle of gldA and regenerate the only SalI site to produce pKP9. PCR using primers SEQ ID NO: 14 and SEQ ID NO: 15 comprising the terminal SalI site, contains kanamycin resistance conferring gene (kan), comprising about 400 bp of upstream translation initiation codon DNA and about 100 bp of downstream translation stop codon The 1.4 kb DNA fragment was isolated from pET-28a (+) (Novagen, Madison, WI) and subcloned into the only SalI site of pKP9 to generate pKP13. 2.1 kb containing the kan insert, starting at 204 bp downstream of the gldA translation initiation codon and ending at 178 bp upstream of the gldA translation stop codon by PCR using primers SEQ ID NO: 16 and SEQ ID NO: 17 comprising terminal SphI and XbaI sites, respectively. DNA fragments were isolated from pKP13 and subcloned between the SphI and XbaI sites in pMAK705 (Genencor International, Palo Alto, Calif.) To generate pMP33. E. coli FM5 was transformed with pMP33 and selected on 20 μg / mL kanamycin at 30 ° C., which is the allowable temperature for pMAK705 replication. At 30 ° C., one colony was grown overnight in liquid medium supplemented with 20 μg / mL kanamycin. Approximately 32,000 cells were plated on 20 μg / mL kan and incubated for 16 hours at 44 ° C., the limit temperature for pMAK705 replication. Transformants grown at 44 ° C. contained plasmids incorporated into the chromosome at a frequency of approximately 0.0001. PCR and Southern blotting [EM Southern, J. Mol. Biol. 98, 503-517 (1975) assays were used to determine chromosomal integrated properties in transformants. Western blotting assays [Towbin et al., Proc. Natl. Acad. Sci. 76, 4350 (1979)] was used to determine whether glycerol dehydrogenase protein, a product of gldA, was produced in the transformants. Activity assays were used to determine whether glycerol dehydrogenase activity remained in the transformants. Conversion of glycerol and NAD ⁺ to dihydroxyacetone and NADH using phenazine methosulfate as the mediator was performed by tetrazolium dye, MTT [3- (4,5-dimethylthiazol-2-yl) -2,5- Diphenyltetrazolium bromide] was coupled to the conversion of dark colored formazan to determine the activity in the glycerol dehydrogenase band on natural gels. Glycerol dehydrogenase also requires 30 mM ammonium sulfate and 100 mM tris, pH 9 [Tang et al., J. Bacteriol. 140, 182 (1997). Six of the eight transformants analyzed were determined to be gldA knockout. Escherichia coli MSP33.6 was deposited with the ATCC on 24 November 1997 under the Budapest Treaty.
[271] Example 4
[272] Construction of Escherichia Coli Strains with KlpK and gld4 Genes Knocked Out
[273] A 1.6 kb DNA fragment containing the gldA gene and comprising 228 bp upstream of the translation initiation codon and 220 bp downstream of the translation stop codon by PCR using primers SEQ ID NO: 18 and SEQ ID NO: 19 containing terminal SphI and XbaI sites, respectively Isolated and cloned between the SphI and XbaI sites of pUC18 to generate pQN2. pQN2 was cleaved the only SalI and NcoI sites in the gldA gene, the ends were clena treated and ligated again to delete 109 bp in the middle of gldA and regenerate the only SalI site to produce pQN4. A 1.2 kb fragment containing the kanamycin resistance conferring gene (kan) and flanked at the loxP site was isolated from pLoxKan2 (Genenco International, Palo Alto, Calif.) As a StuI / XhoI fragment, and the ends were klenowed and cleaved. PQN8 was generated by subcloning into SalI site of poorly treated pQN4. 0.4 kb DNA fragments containing R6K origins of replication were subjected to PCR using primers SEQ ID NO: 20 and SEQ ID NO: 21 containing terminal SphI and XbaI sites, respectively [Miller and Mekalanos, J. Bacteriol. 170, 2575-2583 (1988)] was isolated from pGP704 and ligated to a 2.8 kb SphI / XbaI DNA fragment of pQN8 containing the gldA :: kan cassette to generate pKP22. A 1.0 kb DNA fragment containing the chloramphenicol resistance endowing gene (cam) and flanked at the loxP site was isolated from pLoxCat2 (Genencor International, Palo Alto, CA, USA) as an XbaI fragment, which subsubjected to the XbaI site of pKP22. Cloning produced pKP23. The glpK-E coli strain RJF10 (see Example 2) was transformed with pKP23, the transformants with the phenotype kanRcamS were isolated and showed a double cross integration demonstrated by Southern blot analysis. Glycerol dehydrogenase gel activity assay (described in Example 3) demonstrated that no active glycerol dehydrogenase was present in the transformants. The kan marker was removed from the chromosome using the Cre-producing plasmid pJW168 described in Example 2 to generate strain KLP23. Several isolates with the phenotype kanS demonstrated no glycerol dehydrogenase activity and Southern blot analysis confirmed the loss of the kan marker.
[274] Example 5
[275] Plasmid Construction and Strain Construction for Glycerol 3-Phosphate Dehydrogenase (DAR1) and / or Glycerol 3-Phosphatase (GPP2) Expression
[276] Expression Cassette Preparation for Glycerol 3-Phosphatase (gpp2) :
[277] Saccharomyces cerevisiae chromosome V lambda clone 6592 (Genbank Accession No. # U18813x11) was obtained from ATCC. Cloning from lambda clones using synthetic primers (SEQ ID NO: 22 and SEQ ID NO: 22) comprising the BamHI-RBS-XbaI site at the 5 'end and the SmaI site at the 3' end as the target DNA of the glycerol-3-phosphatase (GPP2) gene did. The product was subcloned into the Srfl site of pCR-Script (Stratagene, Madison, WI) to generate plasmid pAH15 containing GPP2. Plasmid pAH15 contains the GPP2 gene in a direction that is inactive for expression from the lac promoter in pCR-Script SK +. BamHI-SmaI fragment of pAH15 containing GPP2 was inserted into pBlueScriptIISK + to generate plasmid pAH19. pAH19 contains the GPP2 gene in a direction suitable for expression from the lac promoter. The XbaI-PstI fragment of pAH19 containing the GPP2 gene was inserted into pPHOX2 to generate plasmid pAH21. pAH21 / DH5α is an expression plasmid.
[278] Expression Cassette Construction for Glycerol 3-Phosphate Dehydrogenase (DAR1):
[279] DAR1 was isolated by PCR cloning from genomic Saccharomyces cerevisiae DNA using synthetic primers (SEQ ID NO: 24 and SEQ ID NO: 25). Successful PCR cloning into the NcoI site at the 5 'end of DAR1, where ATG in NcoI is DAR1 initiating methionine. The BamHI site is introduced at the 3 'end of DAR1 after the translation terminator. The PCR fragment was digested with NcoI + BamHI and cloned into the same site in the expression plasmid pTrc99A (Pharmacia, Piscatway, NJ) to generate pDAR1.
[280] To produce a better ribosomal binding site at the 5 'end of DAR1, the SpeI-RBS-NcoI linker obtained by annealing the synthetic primers (SEQ ID NO: 26 and SEQ ID NO: 27) was inserted into the NcoI site of pDAR1A to generate pAH40. Plasmid pAH40 contains the novel RBS and DAR1 genes in a direction suitable for expression from the trc promoter of pTrc99A (Parmasia, Piscataway, NJ). A second SpeI-RBS-NcoI linker obtained by annealing the NcoI-BamHI fragment of pDAR1A and the synthetic primers (SEQ ID NO: 28 and SEQ ID NO: 29) was added to the SpeI-BamHI site of pBC-SK + (Stratazine, Madison, WI). Insertion produced plasmid pAH42. Plasmid pAH42 contains the chloramphenicol resistance gene.
[281] Expression cassette construction of dar1 and gpp2 :
[282] Expression cassettes of DAR1 and GPP2 were assembled from DAR1 and GPP2 subclones, respectively, described above using standard molecular biology methods. BAHHI-PstI fragment of pAH19 containing a ribosomal binding site (RBS) and GPP2 gene was inserted into pAH40 to generate pAH43. A BamHI-PstI fragment of pAH19 containing the RBS and GPP2 genes was inserted into pAH42 to produce pAH45.
[283] The ribosomal binding site at the 5 'end of GPP2 was modified as follows. BAHHI-RBS-SpeI linker obtained by annealing the synthetic primers GATCCAGGAAACAGA (SEQ ID NO: 30) and CTAGTCTGTTTCCTG (SEQ ID NO: 31) to the XbaI-PstI fragment of pAH19 containing GPP2 was inserted into the BamHI-PstI site of pAH40 to generate pAH48. Plasmid pAH48 contains the DAR1 gene, modified RBS, and GPP2 gene in a direction suitable for trc promoter expression of pTrc99A (Parmacia, Piscataway, NJ).
[284] E. coli transformation
[285] All plasmids described herein were transformed with E. coli DH5α using standard molecular biology techniques. Transformants were identified by DNA RFLP pattern.
[286] Example 6
[287] Escherichia coli into Klebsiella pneumoniae dhaConstruction of Expression Plasmids for Use in Transformation with Regulon Genes
[288] Expression Vector pTacIQ Construction
[289] PTacIQ, an E. coli expression vector, has been described in pBR322 [Sutcliffe et al., Cold Spring Harb, Symp. Quant. Biol. 43, 77-90 (1979)] lacIq gene [Farabaugh, Nature 274 (5673), 765-769 (1978)] and tac promoter [Amann et al., Gene 25, 167-178] at the endonuclease site EcoRI (1983)]. Multiple cloning sites and termination sequence (SEQ ID NO: 32) replaced the pBR322 sequence from EcoRI to SphI.
[290] Subcloning of the Glycerol Dehydratase Gene (dhaB1, 2, 3, X)
[291] The open reading frame of the dhaB3 gene was amplified from pHK28-26 by PCR using a primer (SEQ ID NO: 33 and SEQ ID NO: 34) containing an EcoRI site at the 5 'end and an XbaI site at the 3' end. The product was subcloned in pLitmus29 (New England Biolab, Inc., Beverly, Mass.) To produce pDHAB3 containing dhaB3.
[292] The region containing the entire coding regions of dhaB1, dhaB2, dhaB3 and dhaBX of the dhaB operon obtained from pHK28-26 was cloned with pBluescriptIIKS + (stratazine from Lazola, Calif.) using restriction enzymes KpnI and EcoRI to make plasmid pM7 Generated.
[293] Plasmid pM7 was cleaved with ApaI and XbaI to remove dhaBX and 5.9 kb fragments were purified and ligated with 325 bp ApaI-XbaI fragment of plasmid pDHAB3 to produce pM11 containing dhaB1, dhaB2 and dhaB3.
[294] The open reading frame of the dhaB1 gene was amplified from pHK28-26 by PCR using primers (SEQ ID NOs: 35 and SEQ ID NO: 36) comprising a HindIII site and a consensus ribosomal binding site at the 5 'end, and an XbaI site at the 3' end. The product was subcloned into pLitmus28 (New England Biolab, Inc.) to produce plasmid pDT1 containing dhaB1.
[295] A NotI-XbaI fragment of pM11 containing a portion of dhaB1 gene, dhaB2 gene and dhaB3 gene was inserted into pDT1 to generate the dhaB expression plasmid, pDT2. A HindIII-XbaI fragment containing the dhaB (1,2,3) gene of pDT2 was inserted into pTacIQ to generate pDT3.
[296] Subcloning of the 1,3-propanediol dehydrogenase gene (dhaT)
[297] The KpnI-SacI fragment of pHK28-26 containing the 1,3-propanediol dehydrogenase (dhaT) gene was subcloned into pBluescriptII KS + to generate plasmid pAH1. The dhaT gene was amplified from pAH1 as template DNA by PCR using synthetic primers (SEQ ID NO: 37 and SEQ ID NO: 38) containing an XbaI site at the 5 'end and a BamHI site at the 3' end. The product was subcloned into the SrfI site of pCR-Script (stratazine) to produce plasmids pAH4 and pAH5 containing dhaT. Plasmid pAH4 contains the dhaT gene in a direction suitable for expression from the lac promoter of pCR-Script, and pAH5 contains the dhaT gene in the opposite direction. The XbaI-BamHI fragment containing the dhaT gene of pAH4 was inserted into pTacIQ to generate plasmid pAH8. HindIII-BamHI fragment of pAH8 containing the RBS and dhaT genes was inserted into pBluescriptIIKS + to generate pAH11.
[298] Expression cassette construction of dhaT and dhaB (1,2,3)
[299] Expression cassettes of dhaT and dhaB (1,2,3) were assembled from the individual dhaB (1,2,3) and dhaT subclones described above using standard molecular biology methods. The SpeI-SacI fragment of pDT3 containing dhaB (1,2,3) was inserted into the SpeI-SacI site of pAH11 to generate pAH24.
[300] PDT16 was constructed by inserting a SalI-XbaI linker (SEQ ID NO: 39 and SEQ ID NO: 40) into pAH5 digested with the restriction enzyme SalI-XbaI. The linker removed the XbaI site. Subsequently, a 1 kb SaII-MluI fragment of pDT16 was inserted into pAH24 to replace the already existing SalI-MluI fragment to generate pDT18. pDT21 was constructed by inserting SalI-NotI fragment of pDT18 and NotI-XbaI fragment of pM7 into pCL1920 (SEQ ID NO: 41). The glucose isomerase promoter sequence (SEQ ID NO: 42) of Streptomyces was cloned by PCR and inserted into the EcoRI-HinDIII site of pLitmus28 to prepare pDT5. pCL1925 was constructed by inserting the EcoRI-PvuII fragment of pDT5 into the EcoRI-PvuI site of pCL1920. pDT24 was constructed by cloning the HinDIII-MluII fragment of pDT21 and the MluI-XbaI fragment of pDT21 to the HinDIII-XbaI site of pCL1925.
[301] Construction of Expression Cassettes of dhaT and dhaB (1,2,3, X) :
[302] pDT21 was constructed by inserting SalI-NotI fragment of pDT18 and NotI-XbaI fragment of pM7 into pCL1920 (SEQ ID NO: 41). The glucose isomerase promoter sequence of Streptomyces (SEQ ID NO: 42) was cloned by PCR and inserted into the EcoRI-HinDIII site of pLitmus28 to prepare pDT5. pCL1925 was constructed by inserting the EcoRI-PvuII fragment of pDT5 into the EcoRI-PvuI site of pCL1920. pDT24 was constructed by cloning the HinDIII-MluII fragment of pDT21 and the MluI-XbaI fragment of pDT21 to the HinDIII-XbaI site of pCL1925.
[303] Construction of expression cassettes of dhaR, orfY, dhaT, orfX, orfW and dhaB (1,2,3, X) :
[304] pDT29 was prepared by inserting the SacI-EcoRI fragment of pHK28-26 into the SacI-EcoRI site of pCL1925.
[305] Construction of expression cassettes of dhaR, orfY, orfX, orfW and dhaB (1,2,3, X) :
[306] Derivatives of plasmid pDT29 were made by deleting the entire gene dhaT except for the first 5 and the last 5 codons (and stop codons) by a technique known as PCR mediated overlap extension. Two main PCR products were obtained using pDT29 as a template and the following primers.
[307] SEQ ID NO: 43 = 5'GAC GCA ACA GTA TTC CGT CGC3 ';
[308] SEQ ID NO: 44 = 5'ATG AGC TAT CGT ATG TTC CGC CAG GCA TTC TGA GTG TTA ACG3 ';
[309] SEQ ID NO: 45 = 5'GCC TGG CGG AAC ATA CGA TAG CTC ATA ATA
[310] TAC3 ';
[311] SEQ ID NO: 46 = 5'CGG GGC GCT GGG CCA GTA CTG3 '.
[312] SEQ ID NO: 45 was paired with SEQ ID NO: 46 to obtain a 931 bp product spanning a nucleic acid comprising 5 'dhaB1 (up to the only ScaI site), all of orfY, and the first five codons of dhaT. SEQ ID NO: 43 was paired with SEQ ID NO: 44 to obtain a product of 1348 bp across the nucleic acid comprising the last five codons (and stop codons) of dhaT, all of orfX, all of orfW, and 5'dhaR (to the only SapI site). The 15 bases at the 5 'end of SEQ ID NO: 44 constitute a complementary tail in the opposite direction to the 15 base portion of SEQ ID NO: 45. Similarly, the 11 bases at the 5 'end of SEQ ID NO: 45 constitute a complementary tail in the opposite direction to the 11 base portion of SEQ ID NO: 44. Thus, the two main PCR products were annealed (by 26 bp tail overlap) and stretched by PCR to connect to each other to yield a 2253 bp third nucleic acid product. The third PCR product was cleaved with SapI and ScaI and ligated to pDT29, which was also cleaved with SapI and ScaI, to produce the same plasmid pKT32 as pDT29 except for a large deletion in frame within dhaT.
[313] Example 7
[314] Conversion of glucose to 1,3-propanediol using E. coli strain KLP23 / pAH48 / pDT29 and improved method using KLP23 / pAH48 / pKP32
[315] Preculture :
[316] 2YT medium containing 10 mg / L carbenicillin (or ampicillin) and 50 mg / L spectinomycin (10 g / L yeast extract, 16 g /) by preincubating KLP23 / pAH48 / pDT29 and KLP23 / pAH48 / pKP32 L tryptone, and 10 g / L NaCl) were inoculated with the fermentor. KLP23 / pAH48 / pKP32 is identical to KLP23 / pAH48 / pDT29 except that dhaT is deleted.
[317] Cultivation was started with frozen stock (10% DMSO as antifreeze) in 500 mL of medium in a 2 L Ellenmeyer flask and incubated on a 35 ° C. shaker 250 rpm to inoculate into the fermenter when approximately 1.0 AU at OD ₅₅₀ was reached.
[318] Fermenter Medium :
[319] The following components was then sterilized together with the fermenter _{vessel: 45 g KH 2 PO 4,} 12 g citric _{acid, 12 g MgS0 4 · 7H 2} 0, 30 g of yeast extract, 2.0 g citrate, ammonium iron (III), 5 mL Mazu DF204 as a Posay , 1.2 g CaCl ₂ · 2H ₂ O, and 7.3 mL sulfuric acid. The pH was raised to 6.8 with 20-28% NH ₄ 0H and the following ingredients were added: 1.2 g carbenicillin or ampicillin, 0.30 g spectinomycin, 60 mL trace element solution and glucose (from 60-67 wt% feed) ). After inoculation, the volume was 6.0 L and the glucose concentration was 10 g / L. Trace element solution contained the following components (g / L): citric acid. _{H 2 0 (4.0), MnSO} 4 H 2 O (3.0), NaCl (1.0), FeSO 4 · 7H 2 O (0.10), CoCl 2 · 6H 2 0 (0.10), ZnS0 4 · 7H 2 0 (0.10) , CuSO ₄ · 5H ₂ 0 (0.010), H ₃ BO ₃ (0.010) and Na ₂ MoO ₄ · 2H ₂ 0 (0.010).
[320] Fermentation culture :
[321] A 15 L stirred tank fermenter was prepared with the above medium. The temperature was adjusted to 35 ° C. and the pH was adjusted to 6.8 using aqueous ammonia (20-28 wt%). Set the initial value of the air flow rate (minimum value set between 6 and 12 standard liters / minute) and the stirrer speed (minimum value set between 350 and 690 rpm) so that the OUR value reaches approximately 140 mmol / L / hour of dissolved oxygen (DO ) Control is started. The back pressure was adjusted to 0.5 bar. DO control was set to 10%. Glucose was maintained between 0 g / L and 10 g / L with a 60% or 67% (wt) feed except for some errors. Vitamin B ₁₂ or coenzyme B ₁₂ was added as follows.
[322] Fermentation with KLP23 / pAH48 / pDT29 :
[323] A representative fermentation scheme for the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23 / pAH48 / pDT29 is shown in Table 4. Beginning 3 hours after inoculation, vitamin B ₁₂ (0.075 g / L, 500 mL) was fed at a rate of 16 mL / hour. The yield of 1,3-propanediol was 24% by weight (1,3-propanediol (g) / glucose consumed (g)) and the titer to 1,3-propanediol was obtained at 68 g / L.
[324] Representative fermentation overview for the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23 / pAH48 / pDT29 Time (h) OD ₅₅₀ (AU) DO (%) Glucose (g / L) Glycerol (g / L) 1,3-PD (g / L) 0 0 150 12.9 0.0 0 6 17 80 8.3 3.1 One 12 42 53 2.8 12.5 9 18 98 9 5.7 12.6 32 24 136 11 32.8 12.0 51 30 148 10 12.3 13.3 62 32 152 11 12.5 14.3 65 38 159 11 1.5 17.2 68
[325] Similar results were obtained when vitamin B ₁₂ was fed in equal concentrations at either twice the concentration or as a concentrated mass over the fermentation process. The highest titer obtained was 77 g / L.
[326] Improved fermentation with KLP23 / pAH48 / pKP32 :
[327] A representative fermentation scheme of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23 / pAH48 / pKP32 is shown in Table 5. Beginning 3 hours after inoculation, vitamin B ₁₂ (0.150 g / L, 500 mL) was fed at a rate of 16 mL / hour. After 36 hours, approximately 2 L of fermentation broth was purged to feed glucose continuously. The yield of 1,3-propanediol was 26% by weight (1,3-propanediol (g) / glucose consumed (g)) and the titer to 1,3-propanediol was obtained at 112 g / L.
[328] Representative Fermentation of Conversion of Glucose to 1,3-propanediol (1,3-PD) Using Escherichia Coli Strain KLP23 / pAH48 / pKP32 Time (h) OD ₅₅₀ (AU) DO (%) Glucose (g / L) Glycerol (g / L) 1,3-PD (g / L) 0 0 148 12.8 0.0 0 6 22 84 6.9 3.3 0 12 34 90 9.7 10.4 7 18 66 43 9.3 5.9 24 24 161 9 0.2 2.5 46 30 200 10 0.2 6.0 67 36 212 10 1.2 9.7 88 42 202 2 0.1 15.5 98 48 197 12 1.2 23.8 112
[329] Similar results were obtained when vitamin B ₁₂ was fed in the same way by adding it as a concentrated mass over a 1 / 2-fold concentration or fermentation process. The highest titer obtained was 114 g / L.
[330] Example 8
[331] Manipulation of the Triosphosphate Isomerase Mutant of Escherichia Coli KLP23 to Improve Yield of 1.3-propanediol from Glucose
[332] Plasmid Construction for Triosphosphate Isomerase Gene Replacement in Escherichia Coli KLP23:
[333] E. coli KLP23 genomic DNA was prepared using a fused DNA isolation kit (Gentra Systems, Minneapolis, Minn.). A 1.0 kb fragment containing the 3 'end of the triosphosphate isomerase (tpiA) gene was PCR from KLP23 genomic DNA using primer sequences 47 and 48 [Mullis and Faloona, Methods Enzymol. 155, 335-350 (1987). 1.0 kb DNA fragments containing the 5 'end of tpiA, yiiQ, and the 5' end of yiiR gene were amplified by PCR from KLP23 genomic DNA using primers SEQ ID NO: 49 and SEQ ID NO: 50. The ScaI site was included in primer sequence 49. The 5 'end of primer sequence 49 was complementary in the opposite direction to primer sequence 48 so that continuous overlap extension PCR could be performed. 2.0 kb fragment by PRR using the two PCR fragments and primer sequences 47 and 50 as a template using gene splicing by the overlap extension technique [Horton et al., BioTechniques 8, 528-535 (1990)] Generated. The fragment showed 73% deletion of the 768 bp tpiA structural gene. Overall, the fragment had a 1.0 kb flanking region on one side of the ScaI cloning site (in part of the tpiA) so that the chromosomal gene could be replaced by homologous recombination.
[334] The blunt terminal 2.0 kb PCR fragment was cloned into the pCR-Blunt vector using a Zero Blunt PCR cloning kit (Invitrogen, San Diego, Calif.), Containing kanamycin and zeocin resistance genes. 5.5 kb plasmid pRN106-2 was generated. ScaI-cleavage using a 1.2 kb HincII fragment of pLoxCat1 (unpublished results) containing a chloramphenicol-resistant gene flanking the bacteriophage P1 loxP site [Snaith et al., Gene 166, 173-174 (1995)] The plasmid pRN106-2 was ligated and inserted into the tpiA fragment in the plasmid pRN106-2 to generate a 6.8 kb plasmid pRN107-1.
[335] Manipulation of triosphosphate isomerase mutant RJ8m by linear DNA transformation :
[336] Using pRN107-1 and primer sequences 47 and 50 as templates, 3.2 kb fragments containing the tpiA flanking region and the loxP-CmR-loxP cassette were PCR amplified and extracted from the gel. E. coli KLP23 was electrotransformed into 1 μg or less of the 3.2 kb linear DNA fragment and glucose on 1 mM glucose in chloramphenicol resistant (12.5 g / mL) and kanamycin sensitive (30 μg / mL) transformants. Transformants that do not have the ability to use normal gluconate on 1 mM gluconate were screened in M9 minimal medium and confirmed that host KLP23 had a phenotype that did not utilize glycerol on 1 mM glycerol. Southern analysis with complete tpiA gene [Southern, J. Mol. Biol. 98, 503-517 (1975), said one mutant, the EcoRI fragment of the RJ8m genomic DNA, showed two 6.6 kb and 3.0 predicted due to the presence of additional EcoRI sites in the chloramphenicol resistance gene. The kb band was observed, indicating that it is a double cross inclusion (tpiA gene substituent). As expected, the host KLP23 and wild type FM5 controls produced single bands of 8.9 kb and 9.4 kb, respectively. The tpiA mutant was further analyzed by genomic PCR using primers SEQ ID NO: 51 and SEQ ID NO: 52 to produce the predicted 4.6 kb PCR fragment, while the host KLP23 and wild type FM5 strains for the same primer pair were both predicted 3.9 kb PCR. Generated a fragment No activity was observed in RJ8m when tpiA activity was tested using glyceraldehyde 3-phosphate as a substrate a cell-free extract from mutant RJ8m and host KLP23. The tpiA mutant RJ8m was electrotransformed with plasmid pAH48 to produce glycerol from glucose, and both plasmids pAH48 and pDT29 or electrotransformed to pKP32 to produce 1,3-propanediol from glucose. Chloramphenicol resistant markers were removed from RJ8m to give RJ8.
[337] Example 9
[338] Conversion of glucose to 1,3-propanediol using E. coli strain RJ8 / pAH48 / pDT29 and improved method using RJ8 / pAH48 / pKP32
[339] Preculture :
[340] RJ8 / pAH48 / pDT29 and RJ8 / pAH48 / pKP32 were preincubated as described in Example 7 for inoculation into fermenters. RJ8 / pAH48 / pKP32 was identical to RJ8 / pAH48 / pDT29 except that dhaT was deleted.
[341] Fermenter Medium :
[342] Fermentor broth is described in Example 7.
[343] Fermentation culture :
[344] Set the initial value of the air flow rate (minimum value set between 5 and 6 standard liters / minute) and the stirrer speed (minimum value set between 300 and 690 rpm) so that the OUR value reaches approximately 60-100 mmol / L / hour The fermenter was incubated as described in Example 7 except for initiating oxygen (DO) control. Vitamin B ₁₂ or coenzyme B ₁₂ was added as follows.
[345] Fermentation using RJ8 / pAH48 / pDT29 :
[346] A representative fermentation scheme for the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain RJ8 / pAH48 / pDT29 is shown in Table 6. Vitamin B ₁₂ was added at 2, 8 and 26 hours in 2, 16 and 16 mg concentrates, respectively. The yield of 1,3-propanediol was 35% by weight (1,3-propanediol (g) / glucose consumed (g)) and the titer to 1,3-propanediol was obtained at 50.1 g / L.
[347] Representative Fermentation Overview for Conversion of Glucose to 1,3-propanediol (1,3-PD) Using Escherichia Coli Strain RJ8 / pAH48 / pDT29 Hours (h) OD ₅₅₀ (AU) DO (%) Glucose (g / L) Glycerol (g / L) 1,3-PD (g / L) 0 0 140 10.6 0.1 0.0 6 5 107 11.1 0.5 0.4 10 16 90 8.5 1.7 1.3 14 25 86 1.8 2.4 5.9 19 38 53 3.5 5.9 15.4 25 53 38 0.1 9.2 26.7 31 54 10 4.5 7.4 39.0 37 37 23 17.2 6.0 45.0 43 21 13 9.9 7.7 50.1
[348] Improved fermentation using RJ8 / pAH48 / pKP32 :
[349] A representative fermentation scheme for the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain RJ8 / pAH48 / pKP32 is shown in Table 7. Vitamin B ₁₂ was added in 48 and 16 mg concentrates at approximately 26 and 44 hours, respectively. The yield of 1,3-propanediol was 34% by weight (1,3-propanediol (g) / glucose consumed (g)) and the titer to 1,3-propanediol was obtained at 129 g / L.
[350] Representative fermentation overview for improved conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain RJ8 / pAH48 / pKP32 Time (h) OD ₅₅₀ (AU) DO (%) Glucose (g / L) Glycerol (g / L) 1,3-PD (g / L) 0 0 150 12.6 0.1 0 6 12 113 6.0 2.6 0 12 24 99 0.0 10.6 0 18 51 76 2.4 28.9 0 24 78 82 2.4 44.2 5 30 114 70 3.8 26.9 33 36 111 72 0.0 20.0 57 42 139 65 0.1 21.9 69 48 157 36 0.1 22.4 79 55 158 25 0.2 21.4 94 64 169 14 0.1 15.8 113 72 169 12 0.1 13.4 119 74 162 14 0.1 14.8 129
[351] Example 10
[352] Identification of Escherichia Coli Nonspecific Catalytic Activity (yqhD) in Improved 1,3-propanediol Method
[353] Demonstration of nonspecific catalytic activity in 1,3-propanediol-producing fermentation with improved catalyst :
[354] Using intact cellular assays for 1,3-propanediol dehydrogenase activity, nonspecific catalytic activity in Escherichia coli is determined by fermentation conditions after addition of vitamin B ₁₂ and the production of 3-hydroxypropionaldehyde (3-HPA). It was proved to exist and not exist before. 10 L of a recombinant E. coli strain containing essentially glycerol-producing plasmid (pAH48) and 1,3-propanediol-producing plasmid (pKP32), except that vitamin B ₁₂ was absent Cultured in fermenter. When the OD ₅₅₀ of the tank reached approximately 100, vitamin B ₁₂ concentrate (48 mg) was added. Immediately before and 2 hours after the addition of vitamin B ₁₂ , an aliquot of the cell was taken from the tank. Cells were recovered by centrifugation and resuspended in original volume in PBS buffer containing 150 μg / mL of chloramphenicol, which inhibits new protein synthesis. Appropriate volumes of chloramphenicol treated cells were added to the reaction mixture (PBS buffer containing 10 g / L glucose, 10 g / L glycerol, 1 mg / L coenzyme B ₁₂ , and 150 μg / mL chloramphenicol). This was added to a 250 mL baffled flask containing an OD ₅₅₀ of 10 and a final volume of approximately 50 mL. The light-blocked flask was shaken at 35 ° C. at 250 rpm. Aliquots for HPLC analysis were taken every hour. 3-HPA production over time was observed in flasks containing cells recovered from the presentation bath prior to or after addition of vitamin B ₁₂ . On the contrary, significant amounts of 1,3-propanediol were observed only in flasks containing cells recovered from fermenters after the addition of vitamin B ₁₂ .
[355] Detection of nonspecific catalytic activity in cell-free extracts :
[356] Natural gel activity staining assays were used to demonstrate nonspecific catalytic activity in cell free extracts. Cells were recovered before and after addition of vitamins from a representative 10 L fermenter cultured with recombinant E. coli strains containing glycerol producing plasmid (pAH48) and 1,3-propanediol producing plasmid (pKP32). Cell-free extracts were prepared by disrupting cells with a French press. Cell-free extracts, pure Klebsiella pneumoniae 1,3-propanediol dehydrogenase (dhaT) formulations, and molecular weight standards were applied and electrophoresed on natural gradient polyacrylamide gels. The gel was then exposed to the substrates 1,3-propanediol and NAD ⁺ , or ethanol and NAD ⁺ . As expected, in the gel when the substrate was 1,3-propanediol, an active dye for DhaT was observed which migrated to approximately 340 KDa on the natural gel. This activity was observed only in lanes to which pure Klebsiella pneumoniae 1,3-propanediol dehydrogenase was applied. In contrast, nonspecific catalytic activity was observed at approximately 90 KDa when the substrate was 1,3-propanediol and then acellular extracts added with vitamin B ₁₂ were applied. When using ethanol as the substrate, neither the DhaT band nor the nonspecific catalytic activity band appeared, but distinct bands appeared at approximately 120 KDa before and after the addition of vitamin B ₁₂ . As typically found in all organisms, this new band will be an alcohol dehydrogenase that is specific for ethanol as a substrate.
[357] The natural gel assay, which separates proteins according to their molecular weight prior to performing the enzymatic analysis step on proteins, is characterized by an alcohol dehydrogenase that is well characterized for E. coli and specific for ethanol as a substrate found in all organisms. It further increases sensitivity and accuracy in measuring the reduction of 1,3-propanediol in such low activity structures that may differ. Dehydrogenase assays operate on the principle that dehydrogenase catalyzes the transfer of electrons from 1,3-propanediol (or other alcohols) to NAD ⁺ . PMS (phenazine methosulfate) is then used to form electrons between NADH and tetrazolium bromide dye (MTT, 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide). The transfer is coupled to form a precipitate in the gel. The gel was soaked in the substrate for several hours or after overnight soaking to remove reagents and soluble dyes. An insoluble blue color was formed in the band with active dehydrogenase on the gel. Various aspects of the assay are described in Johnson and Lin, J. Bacteriol. 169: 2050 (1987).
[358] Purification and Identification of Nonspecific Catalytic Activity in E. Coli:
[359] As described in Example 7, Improved Methods Using KLP23 / pAH48 / pKP32, the nonspecific catalytic activity was partially purified in large quantities on the harvested cells after the completion of the typical 1,3-propanediol production run. Cell pellets (16 g) were washed and resuspended (20 times) in 20 mL of 50 mM Hepes buffer (pH 7.5). Cells in suspension were lysed by sonication. Centrifugation (15 min, 20,000 × g, 10 ° C.) afforded cell-free extracts, which were further clarified by adding 250 mg of protamine sulfate while stirring the supernatant on ice. The supernatant obtained by centrifugation (20 min, 20,000 × g, 10 ° C.) was fractionated by passing through a Superdex® 200 preparative grade column (6 × 60 cm) equilibrated with Hepes buffer. Fractions were collected each 10 mL and each aliquot was concentrated 25 fold using a 10,000 MW Cutoff Centricon® membrane and analyzed by natural gel active staining. Nonspecific catalytic activity was identified in fractions 107-112 and best activity was identified in fractions 108-109. More aliquots of fractions 108 and 109 (each 7 mL each) were concentrated 50-fold and loaded into all lanes of 12-lane natural gels. The gel was cut in half and half stained for dehydrogenase activity, resulting in a dark blue band showing non-specific catalytic activity. The unstained gel was arranged up and down with the stained gel and the band on the undyed gel corresponding to the nonspecific catalytic activity band was cut off. The gel strip was milled, the milled particles were soaked in 0.5 mL of 2D-loading buffer, heated to 95 ° C. for 5 minutes, and centrifuged to remove gel particles to extract soluble protein. Two-Dimensional Polyacrylamide Gel Electrophoresis of Escherichia Coli Extract in a Swiss 2D Database (http://www.expasy.ch/ch2d/; Tonella et al. Electrophoresis 19: 1960-1971 (1998)) (2D-PAGE The supernatant was loaded into an isoelectricfocusing (IEF) strip for 2D-PAGE using the same conditions as described for) The gel was transferred to the PVDF membrane using electroblotting. Colloidal blue gel staining was used to stain the proteins, stained blots used to identify nonspecific catalytic activity are shown in Figure 6. Spots were identified using standard techniques for amino terminal peptide sequencing. The only single spot encoding oxidoreductase activity (spot A) was identified 19. Cycling spot A (FIG. 6) was cycled 19 times and Escherichia coli openley estimated to have oxidoreductase activity by the FASTA search tool. A 100% identical match to the amino-terminus of the dingframe, yqhD was obtained: The complete amino acid sequence for the protein encoded by yqhD is shown in SEQ ID NO: 57; the corresponding DNA sequence is shown in SEQ ID NO: 58. The yqhD gene is Clostridium Sequence identity with adhB, a possible NADH-dependent butanol dehydrogenase 2 gene, is 40%.
[360] Destruction of the gene yqhD in E. coli KLP23 :
[361] Biochemical analysis and amino-terminal sequencing have suggested that nonspecific catalytic activity is encoded by the E. coli ydhD gene. The gene, whose function is unknown, is thought to encode oxidoreductase and is also found in Citrobacter proindei and Klebsiella pneumoniae 1,3-propanediol dehydrogenase encoded by the dhaT gene. It contains two alcohol dehydrogenase signatures.
[362] To destroy this gene, ydhD from genomic DNA and 830 bp 5'- flanking DNA sequence and 906 bp 3'- flanking from PCR using Taq polymerase and primers below DNA sequence was amplified:
[363] (SEQ ID NO: 59) 5'-GCGGTACCGTTGCTCGACGCTCAGGTTTTCGG-3 '
[364] (SEQ ID NO: 60) 5'-GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3 '
[365] After the reaction was run 35 cycles of 1 minute at 94 ° C., 1 minute at 50 ° C., and 3 minutes at 72 ° C., the final stretching was performed at 72 ° C. for 5 minutes. The resulting 3.7 Kb DNA fragment was purified, digested with SacI and KpnI and ligated at 16 ° C. for 16 hours with similarly digested pBluescriptII KS (+) from Stratazine. Ligated DNA was used to transform Escherichia coli DH5α (from Gibco / BRL), and the predicted plasmid pJSP29 was LB agar (DIFCO) containing X-gal (40 μg / mL) and ampicillin (100 μg / mL). Product) from transformants demonstrated as white colonies. Plasmid pJSP29 was cleaved with AflII and NdeI to release a 409 bp DNA fragment comprising the 363 bp yqhD gene and 46 bp 3′- flanking DNA sequence. The remaining 5,350 bp DNA fragment was purified and ligated at 16 ° C. for 16 hours with 1,374 bp AflII / NdeI DNA fragment containing kanamycin resistance gene from pLoxKan2 (Genenco International, Palo Alto, Calif.). The ligated DNA was used to transform E. coli DH5α, and the predicted plasmid pJSP32-Blue was isolated from transformants selected on LB agar medium containing kanamycin (50 μg / mL). Plasmid pJSP32-Blue was digested with KpnI and SacI to purify the 3865 bp yqhD disruption cassette and similarly digested pGP704 [Miller and Mekalanos, J. Bacteriol. 170: 2575-2583 (1988)] at 16 ° C. for 16 hours. Using ligated DNA, Escherichia coli SY327 [Miller and Mekalanos, J. Bacteriol. 170: 2575-2583 (1988)] and the predicted plasmid pJSP32 was isolated from the transformants selected on LB agar medium containing kanamycin (50 μg / mL). E. coli KLP23 was transformed using plasmid pJSP32 and transformants were selected on LB agar containing kanamycin (50 pLg / mL). Screening 200 kanamycin-resistant transformants, two of them proved to be ampicillin sensitive phenotypes predicted for double cross recombination events generated by replacing the yqhD gene with a yqhD disruption cassette.
[366] yqhD gene disruption was demonstrated by PCR using genomic DNA isolated from these two transformants as a template and using the following sets of primer pairs:
[367] Set # 1:
[368] (SEQ ID NO: 61) 5'-GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3 '
[369] (SEQ ID NO: 62) 5'-CAGCTGGCAATTCCGGTTCG-3 '
[370] Set # 2:
[371] (SEQ ID NO: 63) 5'-CCCAGCTGGCAATTCCGGTTCGCTTGCTGT-3 '
[372] (SEQ ID NO: 64) 5'-GGCGACCCGACGCTCCAGACGGAAGCTGGT-3 '
[373] Set # 3:
[374] (SEQ ID NO: 65) 5'-CCGCAAGATTCACGGATGCATCGTGAAGGG-3 '
[375] (SEQ ID NO: 66) 5'-CGCCTTCTTGACGAGTTCTGAGCGGGA-3 '
[376] Set # 4:
[377] (SEQ ID NO: 67) 5'-GGAATTCATGAACAACTTTAATCTGCACAC-3 '
[378] (SEQ ID NO: 68) 5'-GTTTGAGGCGTAAAAAGCTTAGCGGGCGGC-3 '
[379] The reaction was carried out using a Platinum PCR Supermix containing Expand High Fidelity Polymerase (manufactured by Boehringer Manheim) or Taq polymerase (manufactured by Gibco / BRL). After 35 cycles of 1 minute at 94 ° C., 1 minute at 50 ° C., and 2 minutes at 72 ° C., the final stretching was performed at 72 ° C. for 5 minutes. The resulting PCR product was analyzed by gel electrophoresis in 1.0% (w / v) agarose. The results are summarized in Table 8, demonstrating that the yqhD gene was destroyed in both transformants.
[380] Primer set Expected size (bp) Observed size (bp) yqhD destruction yqhD wild type One 1,200 No product About 1,200 2 1,266 No product About 1,266 3 2,594 No product About 2,594 4 No product 1,189 About 900
[381] yqhD disruption resulted in the deletion of the 3 'end comprising the 46 bp 3'- flanking intergenic DNA sequence of yqhD. This deletion eliminated the 363 bp 3 ′ yqhD coding sequence corresponding to 121 amino acids. The stop codon was 15 bp downstream of the yqhD coding sequence remaining in the kanamycin resistance cassette.
[382] Cotransform E. coli KLP23 (yqhD-) with plasmids pAH48 and pKP32, and transformants containing both plasmids contain ampicillin (100 μg / mL) and spectinomycin (50 μg / mL) Selected on LB agar. Representative transformants were tested for the ability to convert glucose to 1,3-propanediol in a 10 L fermenter with or without vitamin B ₁₂ .
[383] In the production of significant amounts of 1.3-propanediol in E. coli strain KLP23 / pAH48 / pKP32, demonstration of the need for yahD :
[384] E. coli strain KLP23 (yqhD-) / pAH48 / pKP32 was used essentially for the production of 1,3-propanediol as described in Example 7 to test the effect of yqhD destruction on 1,3-propanediol production. Fermentation was performed.
[385] Representative 10 L fermentation results using E. coli strain KLP23 (yqhD- / pAH48 / pKP32) knocked out nonspecific catalytic activity are shown in Table 9. The cell mass and glycerol accumulation of the organism were steadily increased until OD ₅₅₀ exceeded 30 A (10.4 hours) until vitamin B ₁₂ was added. Vitamin B ₁₂ was added to 8 mg concentrated mass at 10.4 hours and then fed continuously at a rate of 1.32 mg / hour. Four hours after the addition of B ₁₂ , the glucose consumption rate decreased, the oxygen utilization decreased, and there was no further increase in optical density. Glucose fermentation was stopped and glucose concentration in the tank accumulated. The highest titer of obtained 1,3-propanediol was 0.41 g / L. Serial dilutions of the cells were plated on agar plates containing ampicillin and spectinomycin to test the viability of the organism. These plates were incubated for 24 hours in an incubator at 30 ° C. There was no live colony from the fermentation of E. coli KLP23 (yqhD-) / pAH48 / pKP32 on the plate (Table 11).
[386] In contrast, in cell suspensions from control tanks without the addition of vitamin B ₁₂ , the cell mass and glycerol accumulation continued to increase until the 10 L tank was full by adding all of the glucose feed solution (Table 10). Viability determination tests on agar plates by serial dilutions of the cell suspension at the end of the fermentation showed that the number of viable cells matched the total cell number estimated by the optical density value (Table 11).
[387] Representative fermentation overview for failure to convert glucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23 (yqhD-) / pAH48 / pKP32 Time (h) OD ₅₅₀ (AU) DO (%) Glucose (g / L) Glycerol (g / L) 1,3-PD (g / L) 0 0.4 150 11.3 0.1 0 2.3 3.0 134 10.7 0.13 0 4.3 10.8 85.0 8.2 1.41 0 8.3 23.1 81.8 0.9 10.0 0 16.3 37.2 149 13.1 21.4 0.41 18.3 47.6 149 18.9 21.6 0.39 20.3 39.6 149 24.4 22.3 0.42 23.8 33.6 149 25.4 22.0 0.41
[388] Representative fermentation overview for the conversion of glucose to glycerol using E. coli strain KLP23 (yqhD-) / pAH48 / pKP32 Time (h) OD ₅₅₀ (AU) DO (%) Glucose (g / L) Glycerol (g / L) 0 0.2 148 9.5 0.06 2.2 2.8 128 8.9 0.13 4.2 10.4 58.5 7.0 1.4 8.2 21.6 57.6 2.7 11.2 16.2 76.8 10.7 0 40.5 20.2 117 10.2 0 52.9 23.7 154 8.5 0 63.9 36.2 239 10.1 0.1 122
[389] Representative fermentation overview on viability on plate at the end of glucose fermentation with E. coli strain KLP23 (yqhD-) / pAH48 / pKP32 in the absence and presence of vitamin B ₁₂Vitamin B ₁₂ Time of completion (h) OD ₅₅₀ (AU) Live count (cfu / mL) absence 36.2 239 2.1E11 existence 23.8 33.6 0 existence 23.8 41.2 0
[390]
[391]
[392]
[393]
[1] The present invention includes a method of bioconverting a carbon source fermentable by a single microorganism to 1,3-propanediol.

权利要求:
Claims (29)
[1" claim-type="Currently amended] Encodes a nonspecific catalytic activity that converts 3-hydroxypropionaldehyde to 1,3-propanediol,
(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO: 57,
(b) an isolated nucleic acid fragment substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO: 57,
(c) an isolated nucleic acid fragment encoding a polypeptide of at least 387 amino acids, having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 57,
(d) Isolated nucleic acid fragments hybridized with (a) under hybridization conditions of 0.1 × SSC, 0.1% SDS, 65 ° C., washed with 2 × SSC, 0.1% SDS and then washed with 0.1 × SSC, 0.1% SDS , And
(e) an isolated nucleic acid fragment complementary to (a), (b), (c), or (d)
Isolated nucleic acid fragment selected from the group consisting of.
[2" claim-type="Currently amended] An isolated nucleic acid fragment as set forth in SEQ ID NO: 58.
[3" claim-type="Currently amended] A polypeptide encoded by the isolated nucleic acid fragment of claim 1.
[4" claim-type="Currently amended] The polypeptide of claim 3, as set forth in SEQ ID NO: 57.
[5" claim-type="Currently amended] A chimeric gene comprising the isolated nucleic acid fragment of claim 1 operably linked to a suitable regulatory sequence.
[6" claim-type="Currently amended] The as was transformed microorganism by use of Claim 5 chimeric gene, bakteo (Citrobacter), Enterobacter bakteo (Enterobacter), Clostridium (Clostridium), keulrep when Ella (Klebsiella), Aero bakteo (Aerobacter), Lactobacillus into sheets ( Lactobacillus , Aspergillus , Saccharomyces , Schizosaccharomyces , Zygosaccharomyces , Pichia , Kluyveromyces , Candida (Candida), Hanse Cronulla (Hansenula), debari Oh, my process (Debaryomyces), non-cor (Mucor), torul rope sheath (Torulopsis), methyl bakteo (Methylobacter), Salmonella (Salmonella), Bacillus (Bacillus), Aero bakteo (Aerobacter), Streptomyces (Streptomyces), and the microorganism selected from the group consisting of Pseudomonas (Pseudomonas).
[7" claim-type="Currently amended] (a) one or more genes encoding polypeptides having dehydratase activity,
(b) one or more genes encoding dehydratase reactivation factors, and
(c) at least one exogenous gene encoding a nonspecific catalytic activity of converting 3-hydroxypropionaldehyde to 1,3-propanediol
Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharin, including the functional dhaT gene encoding 1,3-propanediol oxidoreductase Romais, Szezo Caromyces, Jigosaka Romamys, Pizzia, Cluj Veromaises, Candida, Hansenula, Devarios, Mucor, Torulofsis, Methylobacter, Salmonella, Bacillus, Aerobacter A recombinant microorganism useful for producing 1,3-propanediol, selected from the group consisting of Streptomyces, and Pseudomonas.
[8" claim-type="Currently amended] The method of claim 7, wherein
(a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, and
(b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity
Recombinant microorganism further comprising.
[9" claim-type="Currently amended] The recombinant microorganism of claim 7 or 8, wherein the dehydratase reactivation factor is encoded by orfX and orfZ isolated from dha regulator.
[10" claim-type="Currently amended] The recombinant microorganism of claim 9, wherein the orfX and orfZ are isolated independently from Klebsiella spp., Citrobacter spp., Or Clostridium spp.
[11" claim-type="Currently amended] The method of claim 8,
(a) a first gene encoding a polypeptide having glycerol kinase activity,
(b) a second gene encoding a polypeptide having glycerol dehydrogenase activity, and
(c) a third gene encoding a polypeptide having triosphosphate isomerase activity
Wherein said gene further comprises a set of endogenous genes having mutations that inactivate said gene.
[12" claim-type="Currently amended] The recombinant microorganism according to claim 8 or 11, wherein the recombinant microorganism converts a carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and 1-carbon substrates to 1,3-propanediol.
[13" claim-type="Currently amended] 8. The recombinant microorganism of claim 7, wherein the carbon source selected from the group consisting of glycerol and dihydroxyacetone converts to 1,3-propanediol.
[14" claim-type="Currently amended] The gene of claim 8 or 11, wherein the gene encoding the polypeptide having glycerol-3-phosphate dehydrogenase activity is selected from the group consisting of GPD1, GPD2, GPD3, DAR1, gpsA, GUT2, glpD, and glpABC. Recombinant microorganism.
[15" claim-type="Currently amended] The recombinant microorganism according to claim 8 or 11, wherein the gene encoding the polypeptide having glycerol-3-phosphatase activity is selected from the group consisting of GPP1 and GPP2.
[16" claim-type="Currently amended] The gene according to claim 7, 8 or 11, wherein the gene encoding the polypeptide having dehydratase activity is selected from the group consisting of a gene encoding glycerol dehydratase and a gene encoding diol dehydratase. Recombinant microorganism.
[17" claim-type="Currently amended] 12. The recombinant microorganism according to claim 7, 8 or 11, wherein the gene encoding the polypeptide having dehydratase activity is isolated from Klebsiella spp., Citrobacter spp., Or Clostridium spp.
[18" claim-type="Currently amended] (a) at least one gene encoding a polypeptide having dehydratase activity, (ii) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, and (iii) glycerol- An exogenous gene set consisting of at least one gene encoding a polypeptide having 3-phosphatase activity, and (iv) at least one gene encoding a dehydratase reactivation factor, and
(b) at least one endogenous gene encoding a nonspecific catalytic activity of converting 3-hydroxypropionaldehyde to 1,3-propanediol
Recombinant E. coli, comprising, but not a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity.
[19" claim-type="Currently amended] (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity, and (iii ) exogenous gene set consisting of one or more gene subsets encoding gene products of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and
(b) at least one endogenous gene encoding a nonspecific catalytic activity of converting 3-hydroxypropionaldehyde to 1,3-propanediol
Recombinant E. coli, comprising, but not a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity.
[20" claim-type="Currently amended] The method of claim 19,
(a) a gene encoding a polypeptide having glycerol kinase activity,
(b) a gene encoding a polypeptide having glycerol dehydrogenase activity, and
(c) a gene encoding a polypeptide having triosphosphate isomerase activity
And E. coli, wherein each gene further comprises a set of endogenous genes with mutations that inactivate said gene.
[21" claim-type="Currently amended] (a) under suitable conditions, to produce 1,3-propanediol by contacting the recombinant E. coli of claim 19 or 20 with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and 1-carbon substrates. Steps, and
(b) optionally recovering 1,3-propanediol produced in step (a)
Comprising, 1,3-propanediol biological production method.
[22" claim-type="Currently amended] (a) the recombinant E. coli of claim 19 or 20, or (i) at least one exogenous gene encoding a polypeptide having dehydratase activity, (ii) at least one exogenous gene encoding a dehydratase reactivation factor and (iii) the recombinant E. coli of claim 19 or 20 further comprising at least one endogenous gene encoding a nonspecific catalytic activity of converting 3-hydroxypropionaldehyde to 1,3-propanediol. Contacting with at least one carbon source selected from the group consisting of roxyacetone, and
(b) optionally recovering 1,3-propanediol produced in step (a)
Comprising, 1,3-propanediol biological production method.
[23" claim-type="Currently amended] (a) at least one exogenous gene encoding a polypeptide having dehydratase activity, (ii) at least one exogenous gene encoding a dehydratase reactivation factor, and (iii) 3-hydroxypropionaldehyde Recombinant Escherichia coli comprising at least one endogenous gene encoding sufficient nonspecific catalytic activity to convert to 1,3-propanediol, and lacking a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity, is selected from glycerol and Contacting with a first carbon source selected from the group consisting of dihydroxyacetone and a second carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and 1-carbon substrates, and
(b) optionally recovering 1,3-propanediol produced in step (a)
Comprising, 1,3-propanediol production method.
[24" claim-type="Currently amended] The method of claim 23, wherein the recombinant E. coli
(a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity, and (iii ) exogenous gene set consisting of one or more gene subsets encoding gene products of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and
(b) a gene encoding a polypeptide having glycerol kinase activity, (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (iii) a polypeptide having triosphosphate isomerase activity A set of endogenous genes, each gene having a mutation that inactivates the gene
Further comprising.
[25" claim-type="Currently amended] Vector pDT29 comprising a gene set of dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ as set forth in SEQ ID NO: 1.
[26" claim-type="Currently amended] Vector pKP32. Comprising a set of genes of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ as set forth in SEQ ID NO: 1
[27" claim-type="Currently amended] endogenous with (i) a gene encoding a polypeptide having glycerol kinase activity, and (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, each gene having a mutation that inactivates the gene. Two sets of genes,
(b) at least one exogenous gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity,
(c) one or more exogenous genes encoding polypeptides having glycerol-3-phosphatase activity, and
(d) plasmid pKP32
Recombinant Escherichia coli strain KLP23 comprising a.
[28" claim-type="Currently amended] (i) a gene encoding a polypeptide having glycerol kinase activity, (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (iii) a gene encoding a polypeptide having triosphosphate isomerase activity Recombinant E. coli strain RJ8, wherein each gene comprises a set of three endogenous genes, each of which has a mutation that inactivates said gene.
[29" claim-type="Currently amended] (a) Under appropriate conditions, a recombinant E. coli comprising a dha regulator and without a functional dhaT gene encoding 1,3-propanediol oxidoreductase activity, comprises a group consisting of monosaccharides, oligosaccharides, polysaccharides and 1-carbon substrates. Contacting at least one carbon source selected from, and
(b) optionally recovering 1,3-propanediol produced in step (a)
Comprising, 1,3-propanediol production method.

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CA2733616A1|2001-02-22|

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US20030157674A1|2003-08-21|

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JP4716634B2|2011-07-06|

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CA2733616C|2016-09-27|

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US6514733B1|2003-02-04|

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JP2003507022A|2003-02-25|

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US20060121588A1|2006-06-08|

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BRPI0013315B8|2018-02-27|

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US20060148053A1|2006-07-06|

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CA2380616C|2011-05-24|

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AT335823T|2006-09-15|

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WO2001012833A2|2001-02-22|

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DE60029971D1|2006-09-21|

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CN1379818A|2002-11-13|

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US7504250B2|2009-03-17|

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US20090253192A1|2009-10-08|

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DK1204755T3|2006-12-11|

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EP1204755A2|2002-05-15|

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HK1044963B|2007-01-19|

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US7452710B2|2008-11-18|

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BR0013315A|2002-04-02|

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HK1051055A1|2007-09-28|

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PT1204755E|2006-11-30|

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CA2380616A1|2001-02-22|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-08-18|Priority to US14953499P

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1999-08-18|Priority to US60/149,534

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2000-08-18|Application filed by 메리 이. 보울러, 이 아이 듀폰 디 네모아 앤드 캄파니, 마가렛 에이.혼, 제넨코 인터내셔날 인코포레이티드

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2002-07-12|Publication of KR20020059364A

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2007-12-14|Application granted

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2007-12-14|Publication of KR100785997B1

优先权:

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申请号 | 申请日 | 专利标题

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US14953499P| true| 1999-08-18|1999-08-18|

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US60/149,534|1999-08-18|