method for producing a target substance

专利摘要:
METHOD FOR PRODUCING A TARGET SUBSTANCE. The invention concerns a target substance that is produced by cultivating a bacterium with an ability to produce 2-ketoglutaric acid or a derivative thereof as a target substance, and with an ability to produce xylonic acid from xylose, which is provided by the activity of xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase activity, and ketoglutaric 2-semialdehyde dehydrogenase activity, or at which these activities are better, in a medium containing xylose as a carbon source to produce and accumulate the target substance in the medium , and collecting the target substance from the medium.
公开号:BR112013016373B1
申请号:R112013016373-9
申请日:2012-11-06
公开日:2021-05-18
发明作者:Yousuke Nishio；Youko Yamamoto；Kazuteru Yamada；Kosuke Yokota
申请人:Ajinomoto Co., Inc；
IPC主号:

专利说明:

Technical Field
[0001] The present invention relates to a method for producing a target substance by fermentation, such as L-amino acids, using a microorganism, more precisely a method like this for producing a target substance by fermentation using xylose as a raw material. Fundamentals of Technique
[0002] Methods to produce a target substance by fermentation, such as L-amino acids, using a bacterium include methods of using a wild type bacterium (wild type strain), methods of using an auxotrophic strain derived from a wild type strain, methods of using a metabolic-regulating mutant strain derived from a wild-type strain, such as a multi-drug resistant strain, methods of using a strain with properties of both auxotrophic and metabolic-regulating mutant strain, among others.
[0003] For example, L-glutamic acid is produced mainly by fermentation, using an L-glutamic acid-producing bacterium from the group of so-called coryneform bacteria, which belong to the genus Brevibacterium, Corynebacterium or Microbacterium, or a mutant strain of these ( reference, for example, to the document not associated with the patent 1). As methods to produce L-glutamic acid using other strains, there are known methods that use a microorganism belonging to the genus Bacillus, Streptomyces, Penicillium or similar (reference, for example, to patent document 1), methods that use a microorganism belonging to the genus Pseudomonas , Arthrobacter, Serratia, Candida or similar (reference, for example, to patent document 2), methods using a microorganism belonging to the genus Bacillus, or Aerobacter aerogenes (currently Enterobacter aerogenes) or similar (reference, for example, to the document of patent 3), methods using a mutant strain of Escherichia coli (reference, for example, to patent document 4), among others. Furthermore, methods of producing L-glutamic acid using a microorganism belonging to the genus Klebsiella, Erwinia, Pantoea or Enterobacter are also disclosed (reference, for example, to patent documents 5 to 7).
[0004] In recent years, recombinant DNA techniques are being used in the production of target substances by fermentation. For example, a bacterium's L-amino acid productivity is increased by improving the expression of a gene that encodes an L-amino acid biosynthetic enzyme (Patent Documents 8 and 9), or by improving the uptake of a carbon source in the system. L-amino acid biosynthesis (patent document 10).
[0005] In conventional industrial production of substances by fermentation, saccharides were used as a source of carbon, that is, glucose, fructose, sucrose, molasses, hydrolyzed starch, among others, but these are relatively expensive, and the use of materials biomass raw materials derived from plants and the like have also advanced in recent years.
[0006] Although raw materials include edible portions such as starch, fats and oils, are mainly used as such biomass raw materials to date, it is necessary to change in the future such biomass raw materials by those they include non-edible portions, specifically cellulose, hemicellulose, lignin among others. Cellulose and hemicellulose, as inedible biomass, are converted to pentoses or hexoses by means of a pre-treatment using heat or acid, and a saccharification treatment using a cellulase enzyme, and these can be used as raw materials in fermentation ( patent documents 11 and 12). It is known that if mixed saccharides of such pentoses or hexoses are used as the raw material for the fermentation of amino acid etc., Escherichia coli preferentially assimilates glucose and, as a result, two-step proliferation phenomena were observed ( diauxia), growth retardation, etc. (Document not associated with patents 2 and 3)
[0007] In Escherichia coli, a xylose assimilation pathway comprising xylose isomerase encoded by the xylA gene, and xylulokinase encoded by the xylB gene is known, and it is also known that L-amino acids can be produced from xylose by introducing this pathway in Escherichia coli or Corynebacterium glutamicum (Non-Patent Document 5, Patent Document 13, Non-Patent Document 7).
[0008] It is also reported that Caulobacter crescentus and Haloferax volcanii present a way to convert xylose into 2-ketoglutaric acid by means of xylonic acid in five steps, not using a conventionally known way like this, as described above (Document not associated with the patent 6). Furthermore, examples of expression of this pathway in Escherichia coli are also known (Non-patent document 8, patent document 14). Prior Technique References
[0009] Patent Documents: Patent Document 1: U.S. Patent 3,220,929. Patent Document 2: U.S. Patent 3,563,857. Patent Document 3: Japanese Patent Publication (Kokoku) No. 32-9393. Patent Document 4: Japanese Patent Submitted to the Public Domain (Kokai) No. 5-244970. Patent Document 5: Japanese Patent Public Domain 2000-106869 Patent Document 6: Japanese Patent Public Domain 2000-189169. Patent Document 7: Japanese Patent Submitted to the Public Domain 2000-189175. Patent Document 8: U.S. Patent 5,168,056. Patent Document 9: U.S. Patent 5,776,736. Patent Document 10: U.S. Patent 5,906,925. Patent Document 11: Japanese Patent Submitted to the Public Domain, based on PCT Application (Kohyo) No. 9-507386. Patent Document 12: Japanese Patent Submitted to the Public Domain, based on PCT Application No. 11-506934. Patent Document 13: European Patent 1577396. Patent Document 14: U.S. Patent 7,923,226. Non-Patent Document: Non-Patent Document 1: Akashi K. et al., Amino Acid Fermentation, Japan Scientific Societies Press, pp.195-215, 1986. Non-Patent Document 2: Nichols NN et al., Appl. Microbiol. Biotechnol., July 2001, 56(1-2):120-125. Non-Patent Document 3: Gonzalez, R., Biotechnol. Prog., Jan-Feb 2002, 18(1):6-20. Non-Patent Document 4: Song S. et al., J. Bacteriol., Nov 1997, 179(22):7025-7032. Non-Patent Document 5: Tao H. et al., J. Bacteriol., May 2001, 183 (10):2979-2988. Non-Patent Document 6: Stephens, C. et al., J. Bacteriol., March 2007, 189 (5):2181-2185. Non-Patent Document 7: Gopinath, V. et al., Appl. Microbiol. Biotechnol., July 28, 2011 Non-Patent Document 8: Huaiwei, L et al., Bioresour Technol., August 22, 2011. Invention Summary Objective to be achieved by the invention
[0010] An objective of the present invention is to provide a microorganism that can efficiently produce a target substance, such as L-glutamic acid, in a medium containing xylose, and a method for producing a target substance using a microorganism such as this. Means to Achieve the Goal
[0011] The inventors of the present invention studied the development of a microorganism that reproduces using the pathway of conversion of xylose into 2-ketoglutaric acid, through xylonic acid, with the purpose of developing an amino acid-producing bacteria with an ability to assimilate pentose or hexose in reproduction. As a result, it was observed that a microorganism that expresses such a pathway, as described above, can efficiently assimilate xylose and carry out the present invention.
[0012] The present invention thus relates to the following: (1) A method for producing a target substance, comprising cultivating a bacterium with an ability to produce the target substance in a medium containing xylose, as a carbon source, to produce and accumulate the target substance in the medium, and collect the target substance from the medium, where: the target substance is 2-ketoglutaric acid or a derivative thereof, the bacteria has an ability to produce xylonic acid from xylose, and activity of xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase activity and 2-ketoglutaric dehydrogenase 2-semialdehyde activity were transmitted or improved in the bacteria. (2) The method as described above, in which the enzymatic activities of xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and ketoglutaric 2-semialdehyde dehydrogenase were transmitted or improved in the bacterium by introducing genes encoding the enzymes, respectively, into forms that can be expressed in bacteria. (3) The method as described above, in which the gene encoding each of xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and ketoglutaric 2-semialdehyde dehydrogenase is derived from a microorganism belonging to the genus Caulobacter, Escherichia, Agrobacterium , Herbaspirillum, Actinoplanes, Cupriavidus, Pseudomonas, Zobellia, Thermobacillus, Arthrobacter, Azospirillum, Halomonas, Bacillus or Aspergillus. (4) The method as described above, in which the bacteria can produce xylonic acid from xylose, as a result of each of the following characteristics: (A) xylose dehydrogenase activity, or xylose dehydrogenase activity and xylonolactonase activity were transmitted or enhanced in the bacterium, or (B) the bacterium exhibits glucose dehydrogenase activity that can catalyze a reaction that produces xylonic acid from xylose. (5) The method as described above, in which glucose dehydrogenase uses pyrroloquinoline quinone as a coenzyme, and the bacterium exhibits glucose dehydrogenase activity due to its ability to produce pyrroloquinoline quinone, or is cultured in a medium containing pyrroloquinoline quinone. (6) The method as described above, in which the bacterium can produce xylonic acid from xylose, as a result of being introduced with a gene encoding xylose dehydrogenase, or genes encoding xylose dehydrogenase and xylonolactonase in expressible forms . (7) The method as described above, in which the bacterium was modified in such a way that the activity of 2-ketoglutarate dehydrogenase is reduced. (8) The method as described above, in which the bacteria was further modified in such a way that the succinate dehydrogenase activity is reduced. (9) The method as described above, wherein the bacterium is an enterobacterium or a coryneform bacterium. (10) The method as described above, in which the bacterium is a bacterium that belongs to the genus Pantoea. (11) The method as described above, in which the bacterium is Pantoea ananatis. (12) The method as described above, in which the bacterium is a bacterium that belongs to the genus Escherichia. (13) The method as described above, in which the bacterium is Escherichia coli. (14) The method as described above, in which the bacterium is a bacterium that belongs to the genus Corynebacterium. (15) The method as described above, in which the bacterium is Corynebacterium glutamicum. (16) The method as described above, wherein the derivative 2-ketoglutaric acid is a substance selected from the group consisting of L-glutamic acid, L-glutamine, L-arginine, L-citrulline, L-ornithine, L- proline, putrescine and Y-aminobutyric acid. Brief Description of Drawings
[0013] Figure 1 depicts graphs showing results of a growth complementation test for the strain expressing NXA operon derived from C. crescentus, using a strain deficient with respect to the icd gene. E1-αKG, M9-αKG, M9-Xyl and E1-Xyl represent the M9 minimal medium or the E1 synthetic medium, containing 2-ketoglutaric acid or xylose as a single carbon source, respectively.
[0014] Figure 2 depicts graphs showing results of L-glutamic acid production by culturing the E. coli strain, which produces L-glutamic acid and expresses the E. coli ccrNXA operon.
[0015] Figure 3 depicts graphs showing results of L-glutamic acid production by cultivating a strain expressing the ccrNXA operon, using a strain deficient in the xylose assimilation pathway, characteristic in E. coli as a host .
[0016] Figure 4 depicts graphs showing results of L-glutamic acid production by culturing a strain expressing the ccrNXA operon, using a plasmid type with average copy number.
[0017] Figure 5 depicts graphs showing results of L-glutamic acid production through the cultivation of strains expressing the homologous gene xylD, derived from various types of microorganisms. Atu, Hse, Amis and Aor represent Agrobacterium tumefaciens, Herbaspirillum seropedicae, Actinoplanes missouriensis, and Aspergillus oryzae, respectively.
[0018] Figure 6 depicts graphs showing results of L-glutamic acid production by culturing strains expressing the xylX homolog, derived from various types of microorganisms. Art, Atu, Cne, Zga, Tco and Selo represent Arthrobacter globiformis, Agrobacterium tumefaciens, Cupriavidus necator, Zobellia galactanivorans, Thermobacillus composti, and Pseudomonas elodea, respectively.
[0019] Figure 7 depicts graphs showing results of L-glutamic acid production by culturing strains expressing the xylA homolog, derived from various types of microorganisms. Hbo and Abr represent Halomonas boliviensis and Azospirillum brasilense, respectively. Modalities for Carrying Out the Invention:
[0020] The method of the present invention is a method for producing a target substance, comprising cultivating a bacterium with an ability to produce the target substance, in a medium containing xylose as a carbon source, to produce and accumulate the target substance in the medium , and collect the target substance from the medium, where: the target substance is 2-ketoglutaric acid or a derivative thereof, and the bacterium has an ability to produce xylonic acid from xylose, and the xylonate dehydratase activity, activity of 2-keto-3-deoxyxylonate dehydratase and 2-semialdehyde ketoglutaric dehydrogenase activity were transmitted or improved in the bacteria.
[0021] The target substance is 2-ketoglutaric acid (a-ketoglutaric acid, αKG) or a derivative thereof. Examples of the 2-ketoglutaric acid derivative include L-glutamic acid, L-glutamine, L-arginine, L-citrulline, L-ornithine, L-proline, putrescine and Y-aminobutyric acid.
[0022] The "ability to produce a target substance" means an ability of the bacterium used by the present invention to produce a target substance, to such a degree that the target substance can be collected from the cells or medium when it is grown in the medium, preferably an ability to produce the target substance in a greater amount compared to that obtained with a wild-type strain or an unmodified strain grown under the same conditions. The bacteria may have an ability to produce two or more types of target substances.
[0023] The target substance includes a compound as the target substance in a free form and/or a salt thereof, for example, sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, potassium salt, among others.
[0024] 1. Bacteria of the present invention
[0025] The bacterium of the present invention is a bacterium with an ability to produce xylonic acid from xylose, whose xylonate dehydratase activity, 2-keto-3-deoxyxylonate dehydratase activity, and 2-semialdehyde ketoglutaric dehydrogenase activity have been transferred , or where these activities have been improved.
[0026] The bacterium of the present invention may be a bacterium that is not inherent with xylonate dehydratase activity, 2-keto-3-deoxyxylonate dehydratase activity, and 2-semialdehyde ketoglutaric dehydrogenase activity, but received the information of these enzymatic activities , or a bacterium with these inherent enzymatic activities, in which these enzymatic activities are enhanced.
[0027] The ability to produce xylonic acid from xylose is achieved, for example, by one or both of: 1) providing or enhancing xylose dehydrogenase activity, and 2) possessing glucose dehydrogenase activity that can catalyze the reaction which produces xylonic acid from xylose.
[0028] Examples of microorganism to which the meanings of 1) can be applied include bacteria of the genus Escherichia and coryneform bacteria, and examples of microorganism with the property of 2) include bacteria of the genus Pantoea, among others.
[0029] As for the meanings of 1), in addition to the xylose dehydrogenase activity, the xylonolactonase activity can also be improved.
[0030] Bacteria belonging to these genera will be explained later.
[0031] Xylonic acid produced from xylose is converted to 2-ketoglutaric acid by xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and 2-semialdehyde ketoglutaric dehydrogenase. The pathway in which xylose is converted to 2-ketoglutaric acid by xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and ketoglutaric 2-semialdehyde dehydrogenase is also called the Weimberg pathway (J. Biol. Chem., 236:629-636 ). In this specification, the Weimberg pathway and, in addition to the Weimberg pathway, the pathway in which xylose is converted to xylonic acid by xylose dehydrogenase and/or xylonolactonase, may be collectively referred to as the NXA pathway (unpublished xylose assimilation).
[0032] If a bacterium presents the Weimberg pathway, or this pathway is introduced into a bacterium, this can be determined by measuring the enzymatic activities of xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase and 2-semialdehyde ketoglutaric dehydrogenase in an extract of bacteria, or confirming the assimilation of xylonic acid, which is accumulated in a strain that does not have the Weimberg pathway. Furthermore, whether a bacterium presents the NXA pathway, or this pathway has been introduced into a bacterium, this can be determined by measuring the enzymatic activities of xylose dehydrogenase and/or xylonolactonase, in addition to the aforementioned enzymes. Furthermore, these enzymatic activities can also be determined by measuring the xylonic acid produced from xylose.
[0033] In the present invention, xylonate dehydratase is an enzyme that reversibly catalyzes the following reaction (EC4.2.1.82), and is also called D-xylonate dehydratase, D-xylonate dehydratase, or D-xylonate hydrolase. D-xylonic acid -> 2-dehydro-3-deoxy-D-xylonate + H2O
[0034] Xylonate dehydratase activity can be measured, for example, by mixing a solution of D-xylonic acid and a test sample to allow the reaction, ending the reaction below with the addition of a stop solution comprising aqueous solution at 1% semicarbazide hydrochloride and a 1.5% aqueous solution of sodium acetate, and measuring the absorbance of a reaction solution diluted at 250 nm (Dahms, AS, et al., Methods Enzymol., 1982, 90 Pt E:302-5).
[0035] In the present invention, 2-keto-3-deoxyxylonate dehydratase (2-keto-3-deoxy-xylonate dehydratase) is an enzyme that reversibly catalyzes the following reaction (EC4.2.1-). 2-Dehydro-3-deoxy-D-xylonate -> 2-oxoglutaric semialdehyde + H2O
[0036] The activity of 2-keto-3-deoxyxylonate dehydratase can be measured, for example, by mixing a solution of 2-keto-3-deoxyxylonic acid as the substrate and a test sample to enable the reaction, and measuring thereafter the decrease of 2-keto-3-deoxyxylonic acid.
[0037] In the present invention, 2-semialdehyde ketoglutaric dehydrogenase is an oxidoreductase that reversibly catalyzes the following reaction (EC1.2.1.26). 2-Oxoglutaric semialdehyde + NAD(P) -> 2-oxoglutaric acid + NAD(P)H
[0038] The activity of ketoglutaric 2-semialdehyde dehydrogenase can be measured, for example, by measuring the reduction of NAD(P). For example, the activity of this enzyme can be measured by adding ketoglutaric 2-semialdehyde to a mixture of phosphoric acid (pH 8.5), NAD(P) and a test sample, and measuring the absorbance of the reaction mixture at 340 nm ( Adams, E., et al., J. Biol. Chem., 1967, 242, 1802-1814).
[0039] Xylose dehydrogenase (D-xylose-1-dehydrogenase) is one of the dismutases for a pentose and glucuronic acid, and is an oxidoreductase that reversibly catalyzes the following reaction (EC1.1.1.175). D-Xylose + NAD(P)+ -> D-xylonolactone + NAD(P)H + H+
[0040] D-xylose-1-dehydrogenase activity can be measured, for example, by mixing xylose, a test sample and NAD(P) to enable the reaction, and measuring the absorbance of the reaction mixture at 340 nm (Stephens , C. et al., J. Bacteriol., 2007, 189(5):181-2185).
[0041] Xylose dehydrogenase from Caulobacter crescentus can catalyze the reaction that converts D-xylose to xylonic acid.
[0042] In the present invention, xylonolactonase is an enzyme that reversibly catalyzes the following reaction (EC3.1.1.68). D-xylonolactone -> D-xylonic acid
[0043] Xylonolactonase activity can be measured, for example, by mixing xylonolactone and a test sample to allow the reaction, and quantifying the remaining xylonolactone, according to the hydroxamate method (Appl. Microbiol. Biotechnol., 29:375 -379, 1988; Appl. Microbiol., Biotechnol., 27:333-336, 1988).
The genes encoding the enzymes, xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and ketoglutaric 2-semialdehyde dehydrogenase, may be those of any microorganism with the Weimberg pathway and examples include, for example, genes derived from a microorganism such as a bacterium belonging to the genus Caulobacter, Escherichia, Agrobacterium, Herbaspirillum, Actinoplanes, Cupriavidus, Pseudomonas, Zobellia, Thermobacillus, Arthrobacter, Azospirillum, Halomonas, Bacillus, or filamentous fungi belonging to the genus Aspergillus.
[0045] Examples of Caulobacter bacteria include Caulobacter crescentus.
[0046] Like Caulobacter crescentus, strain CB-15 and strain CB-13 are known and stored in the American Type Culture Collection (Address: PO Box 1549, Manassas, VA 20108, United States of America) as ATCC 19089 and ATCC 33532, respectively. In addition, the NA-1000 strain (J. Bacteriol., 192:3678-88, 2010) and the K31 strain can also be used.
[0047] The genome sequences of Caulobacter crescentus strains CB15, NA1000, and K31 are recorded as GenBank accession numbers AE005673, CP001340 and CP000927, respectively.
The enzyme genes of the Caulobacter crescentus strains CB15, NA1000, and K31 are registered in GenBank with the following gene symbols. Table 1

In addition, the xylose dehydrogenase gene and the 2-ketoglutaric dehydrogenase gene from Caulobacter crescentus may be referred to as ccrxylB and ccrxylA, respectively.
[0050] Furthermore, examples of enzymes of the NXA pathway (unpublished xylose assimilation) pathway include, in addition to those of bacteria of the genus Caulobacter, for example, xylose dehydrogenase from Hypocrea jecorina (Trichoderma ressei) (FEMS Microbiol. Lett., 277). , 249-254, 2007); ycbD from Bacillus subtilis (2-ketoglutaric semialdehyde dehydrogenase, type III); 2- ketoglutaric semialdehyde dehydrogenase (type II) from Pseudomonas putida; 2- Ketoglutaric semialdehyde dehydrogenase, type I, type II, type III from Azospirillum brasilense (J. Bac. Chem., 282, 6685-6695, 2007 for these) and their homologues.
In particular, as well as the xylonate dehydratase gene, the yjhG gene and the yagF gene from a bacterium belonging to the genus Escherichia, such as Escherichia coli, can be used. The yjhG gene of Escherichia coli is shown in SEQ ID NO: 34, and the yagF gene of Escherichia coli is shown in SEQ ID NO: 36. In addition, as for xylonate dehydratase, the xylD gene homologues of microorganisms belonging to the genus Agrobacterium, Herbaspirillum, Actinoplanes, or Aspergillus, such as Agrobacterium tumefaciens, Herbaspirillum seropedicae, Actinoplanes missouriensis, and Aspergillus oryzae, can also be used.
[0052] Furthermore, as for 2-keto-deoxyxylonate dehydratase, the xylX gene homologues of bacteria belonging to the genus Agrobacterium, Pseudomonas, Zobellia, Thermobacillus, or Arthrobacter, such as Agrobacterium tumefaciens, Cupriavidus necator, Pseudomonas elodea, Zobellia galactanivoran , Thermobacillus composti, and Arthrobacter globiformis, can also be used.
[0053] In addition, as for ketoglutaric 2-semialdehyde dehydrogenase, the genes of bacteria belonging to the genus Azospirillum, Halomonas, or Bacillus, such as the homologues of the xylA gene of Azospirillum brasilense and Halomonas boliviensis and ycbD of Bacillus subtilis, can also be used.
[0054] The nucleotide sequences of the aforementioned genes and the amino acid sequences encoded by them are shown in table 11.
[0055] In Caulobacter crescentus, the genes of the five enzymes of the NXA pathway constitute an operon structure as described above. The nucleotide sequence of this operon is registered in GenBank as accession number AAK22808_Caulobacter_crescentus. The nucleotide sequence of this operon is shown in SEQ ID NO: 23. The amino acid sequences of 2-keto-3-deoxyxylonate dehydratase, 2-ketoglutaric dehydrogenase, xylose dehydrogenase and xylonolactonase encoded by this operon are shown in SEQ ID NOS: 24 to 27, respectively. In addition, the nucleotide sequence of the xylD gene in this operon, and the xylonate dehydratase amino acid sequences encoded by it are shown in SEQ ID NOS: 28 and 29, respectively. The nucleotide sequence of SEQ ID NO:28 corresponds to positions 5,509 to 7,296 of the sequence of SEQ ID NO:23.
[0056] Although two sites are suggested as the start codon of xylX, positions 1175 to 1177 are described as the start codon in SEQ ID NO: 23. Two start codons are also suggested for xylD, and positions 1 to 3 or as positions 13 to 15 of SEQ ID NO: 28 can be used as the start codon. When positions 13 to 15 are taken as the start codon, the xylonate dehydratase amino acid sequence of SEQ ID NO: 29 starts from Leu of position 5.
[0057] Although glucose dehydrogenase is originally an enzyme that reversibly catalyzes the following reaction (EC1.1.1.119), "glucose dehydrogenase that can catalyze the reaction that produces xylonic acid from xylose" means an enzyme that can convert D-xylose to D-xylonolactone using pyrroloquinoline quinone as a coenzyme. β-D-Glucose + NADP -> D-glucone-1,5-lactone + NADPH + H+
[0058] The pyrroloquinoline quinone can be produced by a capacity that the microorganism originally possesses, or it can be added to the medium (Appl. Environ. Microbiol., 2009 May, 75(9) 2784-2791).
[0059] In the present invention, as well as bacteria that produce 2-ketoglutaric acid or a derivative thereof, it is desirable that the decomposition by means of 2-ketoglutaric acid is attenuated or eliminated. As well as attenuation or elimination of decomposition by means of 2-ketoglutaric acid, it is desirable that the activities or activity of α-ketoglutarate dehydrogenase and/or succinate dehydrogenase be (be) attenuated or eliminated. In the present invention, the activity of α-ketoglutarate dehydrogenase (hereinafter also referred to as "α-KGDH") means an activity of catalyzing the reaction by oxidatively decarboxylating α-ketoglutaric acid (2-oxoglutaric acid) to generate succinyl -CoA. The aforementioned reaction is catalyzed by three types of enzymes, α-KGDH (E1o, α-ketoglutarate dehydrogenase, EC: 1.2.4.2), diiodrolipoamide S-succinyltransferase (E2o, EC: 2.3.1.61), and diiodrolipoamide dehydrogenase (E3, EC:1.8.1.4). That is, these three types of subunits catalyze the following reactions, respectively, and the activity to catalyze a reaction that consists of a combination of these three types of reactions is called the α-KGDH activity. α-KGDH activity can be confirmed by measurements according to the method of Shiio et al. (Isamu Shiio and Kyoko Ujigawa-Takeda, Agric. Biol. Chem., 44 (8), 1897-1904, 1980). E1o: 2-oxoglutarate + [dihydrolipolysin residue succinyltransferase] lipolysin = [dihydrolipolysin residue succinyltransferase] S-succinyldihydrolipolysin + CO2 E2o: CoA + enzyme N6-(S-succinyldihydrolipoyl)lysine = succinyl-dihydro-(N6 enzyme) )lysine E3: N6-(dihydrolipoyl)lysine + NAD+ protein = N6-(lipoyl)lysine + NADH + H+ α-KGDH protein is also called oxoglutarate dehydrogenase or 2-oxoglutarate dehydrogenase.
[0060] In bacteria of the Enterobacteriaceae family, such as Pantoea ananatis, the protein subunits with these three types of enzymatic activities, respectively, form a complex. The subunits are encoded by the sucA, sucB, and lpd genes, respectively, and the sucA and sucB genes exist downstream of the iron-sulfur succinate dehydrogenase (sdhB) protein gene (U.S. Patent 6,331,419). Although these genes are described as the Enterobacter agglomerans AJ13355 genes in the aforementioned patent, this strain was recently reclassified into Pantoea ananatis.
[0061] As well as the genes encoding α-KGDH from enterobacteria, the nucleotide sequences of the sucA gene, the sucB gene and the sucC gene that exist in a downstream region, and the amino acid sequences of the subunits of Pantoea ananatis, are disclosed in European patent application submitted to the public domain 2100957 A1. In addition, the sucA, sucB and sucC genes encoding α-KGDH from Escherichia coli were submitted to the public domain as Genbank NP_415254 and NP_415255, respectively.
[0062] In coryneform bacteria, the E1o subunit is encoded by the odhA gene (registered as NCgl1084 in the GenBank accession number NC_003450, which is also called the sucA gene), and the E3 subunit is encoded by the lpd gene (GenBank accession number Y16642). on the other hand, it is estimated that the E2o subunit is encoded by the odhA gene together with the E1o subunit as a bifunctional protein (Usuda et al., Microbiology, 142, 3347-3354, 1996), or encoded by the gene registered as NCgl2126, in the GenBank accession number NC_003450, which is different from the odhA gene. Therefore, in the present invention, although the odhA gene is a gene that encodes the E1o subunit, it can also encode E2o.
[0063] The nucleotide sequence of the odhA gene of Brevibacterium lactofermentum and the amino acid sequence of the E1o subunit so encoded (WO2006/028298), the nucleotide sequence of NCgl2126 previously mentioned with the GenBank accession number NC_003450, and the sequence of amino acids of the thus encoded E2o subunit, as well as the aforementioned NCg11084 nucleotide sequence with GenBank accession number NC_003450, and the amino acid sequence of the thus encoded E1o subunit are disclosed in European patent application submitted to public domain 2100957 A1.
[0064] In the present invention, the genes encoding each of the α-KGDH subunits, and the gene cluster containing them, may be referred to generically as the "genes encoding α-KGDH".
[0065] Succinate dehydrogenase (hereinafter also referred to as "SDH") is an enzyme of EC:1.3.99.1, which reversibly catalyzes the following reaction. In the present invention, SDH activity means the activity to catalyze this reaction. Succinic acid + FAD -> fumaric acid + FADH2
[0066] SDH is composed of three or four subunit structures that depend on the type of microorganism, and its activity can be reduced or eliminated by modifying at least one of these proteins, in such a way that they do not function normally. Specifically, SDH is composed of the following subunits (the names of the genes encoding the subunits are described in parentheses), and the membrane anchor protein is encoded only by sdhC or by sdhC and sdhD, depending on the species. SDHA: flavoprotein subunit (sdhA) SDHB: Fe-S protein subunit (sdhB) SDHC: membrane anchoring protein (sdhC) SDHD: membrane anchoring protein (sdhD)
[0067] In addition, the SDH subunit complex may exhibit both SDH and fumarate reductase activities. For example, the SDH subunit complex of coryneform bacteria exhibits both SDH and fumarate reductase activities (WO2005/021770).
[0068] The activity of SDH can be confirmed by measuring the reduction of 2,6-dichloroindophenol (DCIP) as an indicative index. A specific method is described in Tatsuki Kurokawa and Junshi Sakamoto, Arch. Microbiol., (2005) 183:317-324.
[0069] In the present invention, the genes encoding the SDH subunits, and the operon containing them, may be generically referred to as the "genes encoding SDH."
[0070] As well as the genes encoding SDH from enterobacteria, the nucleotide sequences of such genes from Pantoea ananatis and the amino acid sequences of the subunits are disclosed in WO2008/075483.
[0071] As well as the genes encoding SDH of coryneform bacteria, for example, the sequences of the sdh operon of Corynebacterium glutamicum (GenBank accession number NCgl0359 (sdhC) NCgl0360 (sdhA) NCgl0361 (sdhB)), and the operon are disclosed sdh of Brevibacterium flavum (Japanese Patent Public Domain 2005-095169, European Patent Application Public Domain 1672077 A1, WO2008/075483).
[0072] To reduce or eliminate the activities of α-KGDH and SDH, recently described methods for reducing the activity of an enzyme that catalyzes a reaction that branches from the L-glutamic acid biosynthesis pathway can be used, and produces other compounds.
[0073] Furthermore, in the microorganism of the present invention, the activity of an enzyme that incorporates xylose into cells can be further improved.
Examples of the enzyme that incorporates xylose into cells include D-xylose permease, and examples of the gene encoding D-xylose permease include the xylE gene. The nucleotide sequence of the Escherichia coli xylE gene encoding D-xylose permease, and the amino acid sequence encoded by this gene are shown in SEQ ID NOS: 30 and 31, respectively.
[0075] Furthermore, it is desirable that, in the microorganism of the present invention, the xylose isomerase (xylA) and xylulose kinase (xylB) are attenuated. The xylose isomerase (xylA) gene and the xylulose kinase (xylB) gene of Escherichia coli are disclosed as NC000913.1 gi:16131436 and 16131435, respectively.
[0076] In the present invention, xylonic acid can be accumulated in the medium and, especially in Escherichia coli, it is desirable that the xylonate dehydratase activity is further improved. For example, it is preferably best in such a way that it shows the activity thereof at 10 µmol/min/mg protein or more, desirably 15 µmol/min/mg protein or more, more desirably 17 µmol/min/mg protein or more.
[0077] Methods to provide the activity of a target enzyme to a microorganism, or to increase the activity of a target enzyme to a microorganism will be explained below.
[0078] When the microorganism does not originally exhibit the activity of a target enzyme, the activity of a target enzyme can be provided to the microorganism by introducing a target enzyme gene into the microorganism. In addition, when the microorganism exhibits target enzyme activity, introducing a foreign target enzyme gene, increasing the copy number of the endogenous target enzyme gene, or modifying an expression control sequence such as the enzyme gene promoter target to increase gene expression, the activity of a target enzyme may be higher. In the present invention, the expression "to introduce a target enzyme gene" means not only to introduce a target enzyme gene into a microorganism that does not originally have the activity of the target enzyme, but also to introduce a foreign target enzyme gene into a microorganism with activity of the target enzyme, and introducing an endogenous target enzyme gene into a microorganism with target enzyme activity to fuel expression of the endogenous target enzyme gene.
[0079] In order to introduce a target enzyme gene, for example, the target enzyme gene is cloned into an appropriate vector, and a host microorganism is transformed with the obtained vector.
[0080] Examples of the vector used for transformation include a plasmid that can autonomously replicate in a microorganism of choice. Examples of plasmids that replicate autonomously in a microorganism belonging to the Enterobacteriaceae family include pUC19, pUC18, pBR322, RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29, pTWV228, pTWV229 (vectors from the pTWV229 and pTWTV series are series available from Takara Bio), pMW119, pMW118, pMW219, pMW218 (pMW series vectors are available from Nippon Gene) among others. In addition, plasmids from coryneform bacteria include pAM330 (Japanese Patent Public Domain 58-67699), pHM1519 (Japanese Patent Public Domain 58-77895), pSFK6 (Japanese Patent Public Domain 2000-262288), pVK7 ( published patent application US 2003/0175912), pAJ655, pAJ611, pAJ1844 (Japanese patent submitted to the public domain 58-192900), pCG1 (Japanese patent submitted to the public domain 57-134500), pCG2 (Japanese patent submitted to the public domain 58- 35197), pCG4, pCG11 (Japanese patent submitted to the public domain 57-183799), pHK4 (Japanese patent submitted to the public domain 5-7491) among others.
[0081] Examples of transformation methods include treating recipient cells with sodium chloride to increase permeability with respect to DNA, which has been reported in Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol Mol. Biol., 1970, 53:159-162), preparing competent cells from cells that are in the growth phase, followed by transformation with DNA, which has been reported in Bacillus subtilis (Duncan, CH, Wilson, GA and Young , FE, 1977, Gene, 1:153-167), among others. Alternatively, a method of preparing the DNA recipient cells in protoplasts or spheroplasts, which can easily capture the recombinant DNA, followed by the introduction of a recombinant DNA into the DNA recipient cells, which is known to be applicable to Bacillus subtilis, actinomycetes and yeast (Chang, S. and Choen, SN, 1979, Mol. Gen. Genet., 168:111-115; Bibb, MJ, Ward, JM and Hopwood, OA, 1978, Nature, 274:398-400; Hinnen, A ., Hicks, JB and Fink, GR, 1978, Proc. Natl. Sci., USA, 75:19291933) may also be employed. Furthermore, the transformation of microorganisms can also be carried out by the electrical pulse method (Japanese Patent Public Domain 2-207791).
[0082] The target enzyme gene can also be introduced by introducing the gene into a chromosome of the host microorganism. The target enzyme gene can be introduced into a chromosome of a microorganism by a method of randomly introducing into a chromosome, using a transposon or Mini-Mu (Japanese Patent Public Domain 2-109985, US patent 5,882,888, publication by European patent 805867 B1), or by homologous recombination using a sequence present in a chromosomal DNA in a multiple copy number as a target. Like a sequence present in chromosomal DNA in a multiple copy number, repetitive DNA located inverted and repeated at the end of a transposer can be used. Alternatively, using the Red-directed integration method (WO2005/010175), it is also possible to introduce a target gene into a chromosome. Furthermore, a target gene can also be introduced into a chromosome by transduction using phages such as P1 phage, or using a conjugative transfer vector. Furthermore, it is also possible to introduce a target enzyme gene using a gene not necessary for the production of target substance as a target, in the manner described in WO03/040373. One or more copies of the target enzyme gene can be introduced into a target sequence by such methods in the manner described above.
[0083] The transfer of a target gene on a chromosome can be confirmed by Southern hybridization, using a probe with a sequence complementary to the target gene or a part of it.
While it is sufficient that the number of copies of the introduced target gene is not less than 1, the copy number is preferably 2 or more, more preferably 3 or more, even more preferably 5 or more. As for the xylonate dehydratase gene as the target gene, in particular, it is preferable to introduce 2 or more copies of the gene.
[0085] Furthermore, the activity of a target enzyme gene can be optimized using substitution or mutation of an expression control sequence, such as a target enzyme gene promoter in combination, in the manner described above. In particular, it is preferable that the xylonate dehydratase gene is overexpressed by substituting or mutating an expression control sequence in place of, or together with, the aforementioned copy number.
[0086] Examples of the method for increasing the expression of a target enzyme gene include replacing an expression control sequence, such as a target enzyme gene promoter, with one with an appropriate gap in a chromosomal DNA or a plasmid to improve gene expression. For example, it is known that the thr promoter, lac promoter, trp promoter, trc promoter, pL promoter, tac promoter, among others, are often used as promoters. In addition, the tac promoter variants used in the examples described below (PtacA promoter, PtacB promoter) can also be used. Methods to assess the range of strong promoters and promoters are described in the paper by Goldstein and Doi (Goldstein, MA and Doi RH, 1995, Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1, 105-128), among others .
[0087] Furthermore, it is also possible to introduce nucleotide substitution for several nucleotides, in a promoter region of a gene, to modify them in a promoter with a suitable gap, in the manner disclosed in the international publication WO00/18935. Expression control sequence replacement can be performed in the same way, for example, as that of gene replacement using a temperature-sensitive plasmid. Examples of vectors with a temperature-sensitive origin of replication and used for Escherichia coli and Pantoea ananatis include, for example, the temperature-sensitive plasmid pMAN997 described in international publication WO99/03988, derivatives thereof, among others. In addition, the replacement of an expression control sequence can also be performed by a method that uses a linear DNA, such as the method termed "Red-directed integration" using phage À Red recombinase. (Datsenko, KA and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA. 97:6640-6645), and the method using a combination of the Red-directed integration method and the À phage excision system . (Cho, E.H., Gumport, R.I., Gardner, J.F., J. Bacteriol. 184: 5200-5203 (2002)) (reference to WO2005/010175). Modification of an expression control sequence can be combined with the highest gene copy number.
[0088] Furthermore, it is known that the substitution of several nucleotides in a spacer between the ribosome binding site (RBS) and the translation start codon, especially a sequence immediately upstream of the start codon, markedly affects the mRNA translation efficiency and therefore this sequence can be modified to improve the amount of translation.
[0089] When a target gene is introduced into the aforementioned amplification plasmid or chromosome, any promoter can be used to express the gene, as long as the chosen promoter works in a microorganism used. The promoter can be the natural promoter for the gene used or a modified promoter. The expression of a gene can also be controlled by appropriately choosing a promoter that acts strongly in a microorganism used, or by making the -35 and -10 regions of the promoter closer to the consensus sequence.
[0090] Whether the activity of a target enzyme is better or not, this can be confirmed by comparing the activity of the target enzyme of a modified strain and a parental or unmodified strain. If the target enzyme activity of the modified strain is higher compared to the parent or unmodified strain, the target enzyme activity is improved. Furthermore, when the parent strain does not have target enzyme activity, if target enzyme activity can be detected in the modified strain, target enzyme activity is improved.
[0091] The target enzyme gene can be obtained by PCR using oligonucleotides prepared based on the aforementioned sequence information, or gene or protein sequence information known by the microorganism as oligonucleotide primers, or hybridization using an oligonucleotide prepared based on the information of the aforementioned sequence as a probe, from a chromosomal DNA or chromosomal DNA library of a microorganism with the target enzyme.
[0092] Furthermore, the target enzyme and the gene encoding it may be a homologue or artificial modification thereof, or a protein with a conservative mutation or a gene encoding the same, as long as the enzyme activity is maintained.
A homologue such as this, the artificial modification thereof, or a protein with a conservative mutation or genes encoding it are referred to as a conservative variant.
[0094] The conservative variant of a target enzyme can be, for example, a protein with the amino acid sequence of each enzyme mentioned above, but which includes substitution, deletion, insertion, addition or the like of one or more amino acid residues in a or multiple positions.
[0095] Although the number of "one or several" amino acid residues may differ depending on the position in the three-dimensional structure or the types of amino acid residues of the protein, specifically, it is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5. A conservative mutation is typically a conservative substitution. Conservative substitution is a mutation, in which the substitution mutually occurs between Phe, Trp, and Tyr, if the substitution site is an aromatic amino acid; between Leu, Ile and Val, if the replacement site is a hydrophobic amino acid; between Gln and Asn, if the substitution site is a polar amino acid; between Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid with a hydroxyl group. Substitutions considered conservative substitution specifically include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp , replacement of Ser or Ala by Cys, replacement of Asn, Glu, Lys, His, Asp or Arg by Gln, replacement of Gly, Asn, Gln, Lys or Asp by Glu, replacement of Pro by Gly, replacement of Asn, Lys , Gln, Arg or Tyr by His, replacement of Leu, Met, Val or Phe by Ile, replacement of Ile, Met, Val or Phe by Leu, replacement of Asn, Glu, Gln, His or Arg by Lys, replacement of Ile , Leu, Val or Phe by Met, replacement of Trp, Tyr, Met, Ile or Leu by Phe, replacement of Thr or Ala by Ser, replacement of Ser or Ala by Thr, replacement of Phe or Tyr by Trp, replacement of His , Phe or Trp by Tyr, and substitution of Met, Ile, or Leu by Val. Amino acid substitutions, deletions, insertions, additions, inversions, or the like mentioned above, may be a result of a naturally occurring mutation or variation, or a variation due to an individual difference or species difference of a microorganism, from which the genes are derived (mutant or variant). Such proteins can be obtained, for example, by modifying a nucleotide sequence of a wild-type target enzyme gene by site-specific mutagenesis, such that amino acid residues at specific sites in the encoded protein include substitutions, deletions, insertions or additions of amino acid residues.
[0096] Furthermore, such a protein with a conservative mutation, in the manner described above, may be a protein that exhibits a homology of, for example, 80% or more, preferably 90% or more, more preferably 95% or more , even more preferably 97% or more, preferably additionally 98% or more, particularly preferably 99% or more, with the total amino acid sequence, and with a function equivalent to that of the wild-type protein. In this specification, "homology" can mean "identity".
[0097] As long as the wild-type target enzyme gene encodes such an amino acid sequence as described above, it is not limited to Caulobacter crescentus, Haloferax volcanii and the like genes, but can be any of those with an equivalent codon to an arbitrary codon.
[0098] The wild-type gene can also be a DNA that is capable of hybridizing to a nucleotide sequence complementary to the nucleotide sequence of each enzyme gene, or a probe that can be prepared from the complementary sequence under stringent conditions, and encodes a protein with functions equivalent to those of the wild-type target enzyme. "Strict conditions" refer to the conditions under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed. Examples of stringent conditions include those in which highly homologous DNAs hybridize to each other, for example, DNAs not less than 80% homologous, preferably not less than 90% homologous, more preferably not less than 95% homologous, even more preferably not less than 97% homologs, preferably additionally not less than 98% homologs, particularly preferably not less than 99% homologs hybridize to each other, and DNAs less homologous than above do not hybridize to each other, or the conditions which correspond to the typical Southern hybridization wash, i.e. one wash conditions, preferably 2 or 3 times, at a salt concentration and temperature of 1 x SSC, 0.1% SDS at 60°C, preferably 0 SSC. 1x, 0.1% SDS at 60°C, more preferably 0.1x SSC, 0.1% SDS at 68°C.
[0099] As with the probe, a part of a sequence that is complementary to a target enzyme gene can also be used. Such a probe can be prepared by PCR using oligonucleotides prepared based on the known sequence of a gene, with oligonucleotide primers and a DNA fragment containing the nucleotide sequence as a template. For example, when a DNA fragment with a size of about 300 bp is used as the probe, the hybridization wash conditions can be, for example, 50°C, 2x SSC and 0.1% SDS.
[00100] The aforementioned descriptions, with respect to conservative variants of the proteins and genes encoding the same aforementioned, are similarly applied to the other genes described below for bacteria producing target substances.
[00101] The microorganism used in the present invention may inherently exhibit an ability to produce a target substance, or the ability may be provided in reproduction using a mutation method, a recombinant DNA technique or the like.
[00102] The microorganisms used in the present invention include, but are not limited to, bacteria belonging to the Enterobacteriaceae family, such as those of the Escherichia, Pantoea, and Enterobacter genera, coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium lactofermentum and bacteria of the Bacillus genus such as Bacillus subtilis.
[00103] In the present invention, coryneform bacteria include those bacteria that were hitherto classified in the genus Brevibacterium, but are currently classified in the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteria that belong to the genus Brevibacterium closely related to the genus Corynebacterium. Examples of such coryneform bacteria are listed below. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (efficiens Corynebacterium) Corynebacterium herculis Brevibacterium divaricatum Brevibacterium flavum Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum Examples Specific bacteria include the following. Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060 Corynebacterium lilium ATCC 15995 14020 Brevibacterium flavum ATCC 13826, ATCC 14067 Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum ATCC 13869 (Corynebacterium glutamicum ATCC 13869) Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 68vibacterium ATCC 19240 Brevibacterium ceribacterium ATCC1271 ATCC 19240 Brevibacterium ceribacterium atmm ammoniaphilum ATCC 15354
[00104] These strains are available from the American Type Culture Collection (ATCC) (Address: P.O. Box 1549, Manassas, VA 20108, 1, United States). That is, a registration number is determined for each of the strains. Strains can be classified using this registration number (http://www.atcc.org/). “Registration numbers assigned to strains listed in the catalog of the ATCC”. Strain AJ12340 was deposited on October 27, 1987 at the National Institute of Bioscience and Human Technology of the Agency of Industrial Science and Technology (now an independent administrative agency, National Institute of Technology and Evaluation, International Patent Organism Depositary, Tsukuba Central 6, 1- 1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) with accession number FERM BP-1539, based on the Budapest Treaty.
Microorganisms belonging to the Enterobacteriaceae family used in the present invention include, but not limited to, bacteria belonging to the genera Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella, Morganella or the like, and with an ability to produce substance target. Specifically, bacteria that belong to the Enterobacteriaceae family, according to the classification shown in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/ wgetorg mode=Tree&id=1236&lvl=3&keep=1&srchmode=1 &unlock), can be used. Among the bacteria of the Enterobacteriaceae family, bacteria belonging to the genus Escherichia, Enterobacter or Pantoea are preferably used as the parent strain.
[00106] Escherichia genus bacteria that can be used as the parent strain include, but are not limited to, Escherichia genus bacteria reported by Neidhardt et al. (Neidhardt, F.C. et al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1029 table 1), such as Escherichia coli. Specific examples of Escherichia coli include the Escherichia coli W3110 strain (ATCC 27325) and the MG1655 strain (ATCC 47076), which is derived from the wild type strain of Escherichia coli K12 (prototype), among others.
[00107] In particular, bacteria of the genera Pantoeas, Erwinia and Enterobacter are classified as Y-proteobacteria, and are taxonomically very close to each other (J. Gen. Appl. Microbiol., Dec. 1997, 43(6), 355- 361; International Journal of Systematic Bacteriology, Oct. 1997, pp.1061-1067). In recent years, some bacteria belonging to the Enterobacter genus have been reclassified as Pantoea agglomerans, Pantoea dispersa or similar, based on DNA-DNA hybridization experiments, etc. (International Journal of Systematic Bacteriology, July 1989, 39(3).p.337-345). In addition, some bacteria belonging to the Erwinia genus have been reclassified as Pantoea ananas or Pantoea stewartii (reference to International Journal of Systematic Bacteriology, January 1993, 43(1), pp.162-173). Furthermore, Pantoea ananas was then further reclassified as Pantoea ananatis.
[00108] Examples of bacteria of the Enterobacter genus include Enterobacter agglomerans (now reclassified as Pantoea ananatis etc.), Enterobacter aerogenes, among others. Specifically, the strains exemplified in public domain European patent application 952221 can be used. A typical strain of the Enterobacter genus is Enterobacter agglomeranses ATCC 12287 (now reclassified as Pantoea ananatis).
[00109] Typical strains of bacteria of the genus Pantoeas include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans and Pantoea citrea. Specific examples include the following strains:
[00110] Pantoea ananatis AJ13355 (FERM BP-6614, European patent application submitted to the public domain 0952221)
[00111] Pantoea ananatis AJ13356 (FERM BP-6615, European patent application submitted to the public domain 0952221)
[00112] Although these strains are described as Enterobacter agglomerans in European patent application submitted to public domain 0952221, they are currently classified as Pantoea ananatis, based on nucleotide sequence analysis of 16S rRNA etc., in the manner described above.
[00113] The strain Pantoea ananatis AJ13355 was isolated from soil in Iwatashi, Shizuoka in Japan, as a strain that can proliferate in a medium containing L-glutamic acid and a carbon source at low pH. Strain SC17 was selected as a mutant strain that produces low viscous substance from strain AJ13355 (U.S. patent 6,596,517). The strain Pantoea ananatis AJ13355 has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently National Institute of Technology and Evaluation, International Patent Organism Depositary, address: Tsukuba Central 6 , 1-1, Higashi 1-Chome, Tsukuba-shi, Ibarakiken, 305-8566, Japan) on February 19, 1998 and assigned an accession number of FERM P-16644. The deposit was then converted to an international deposit under the terms of the Budapest Treaty on January 11, 1999, and is assigned an accession number of FERM BP-6614. The Pantoea ananatis SC17 strain was provided with a proprietary number of AJ416 and deposited on February 4, 2009 at the National Institute of Technology and Evaluation, International Patent Organism Depository (address: Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba -shi, Ibaraki-ken, 305-8566, Japan), and assigned an accession number of FERM BP-11091.
Examples of Pantoea ananatis bacteria that produce L-glutamic acid include strains SC17sucA/RSFCPG+pSTVCB, AJ13601, NP106 and NA1. The strain SC17sucA/RSFCPG+pSTVCB was obtained by introducing the plasmid RSFCPG containing the citrate synthase (gltA) gene, the phosphoenolpyruvate carboxylase (prpC) gene, and the glutamate dehydrogenase (gdhA) gene derived from Escherichia coli, and the plasmid pSTV containing the citrate synthase (gltA) gene derived from Brevibacterium lactofermentum, in strain SC17sucA, which is a strain deficient with respect to the sucA gene, derived from strain SC17 (US patent 6,596,517). Strain AJ13601 was selected from strain SC17sucA/RSFCPG+pSTVCB for its resistance to L-glutamic acid at high concentration, low pH. Furthermore, strain NP106 was derived from strain AJ13601 by deleting plasmid RSFCPG+pSTVCB (WO2010/027045). Strain AJ13601 was deposited with the National Institute of Technology and Evaluation, International Patent Organism Depositary (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, zip code: 305-8566) on 18 of August 1999, and determined with an accession number FERM P-17516. The deposit was then converted to an international deposit under the terms of the Budapest Treaty on July 6, 2000, and assigned an accession number FERM BP-7207. This strain was originally identified as Enterobacter agglomerans when it was isolated, and deposited as Enterobacter agglomerans. However, it was easily reclassified as Pantoea ananatis, based on 16S rRNA nucleotide sequencing, among others.
[00115] The NA1 strain is a strain that corresponds to the NP106 strain with RSFPPG (WO2008/020654), in which the RSFCPG gltA gene described above is replaced by the methyl citrate synthase (prpC) gene (WO2010/027045).
[00116] Examples of bacteria of the genus Erwinia include Erwinia amylovora and Erwinia carotovora, and examples of bacteria of the genus Klebsiella include Klebsiella planticola. Specific examples include the following strains: Erwinia amylovora ATCC 15580 Erwinia carotovora ATCC 15713 Klebsiella planticola AJ13399 (FERM BP-6600, European patent placed in the public domain 955368) Klebsiella planticola AJ13410 (FERM BP-6617, European patent placed in the public domain 955368) .
[00117] Hereinafter, methods for providing an ability to produce a target substance for such microorganisms in the manner described above, or methods for improving an ability to produce a target substance for such microorganisms, are described.
[00118] To provide an ability to produce a target substance, the methods conventionally employed in the cultivation of coryneform bacteria or bacteria of the genus Escherichia (see “Amino Acid Fermentation”, Gakkai Shuppan Center (Ltd.), 1st Edition, published 30th May 1986, pp. 77-100) can be used. Such methods include the acquisition of an auxotrophic mutant, a strain resistant to the target substance analogue, or a metabolic regulation mutant, construction of a recombinant strain in which the expression of the target substance biosynthesis enzyme is better, among others. During reproduction of bacteria that produce target substance, the properties provided such as an auxotrophic mutation, analogous resistance, or mutation in metabolic regulation may be one or more. The target substance expression biosynthesis enzyme(s) may be better alone or in combinations of two or more. Furthermore, the provision of properties, such as an auxotrophic mutation, analogous resistance or mutation in metabolic regulation, can be combined with improved biosynthesis enzymes.
[00119] An auxotrophic mutant strain, analogous resistant strain, or metabolic regulation mutant strain, with an ability to produce a target substance, can be obtained by subjecting a parental strain or wild-type strain to conventional mutagenesis, such as exposure to X-rays or UV irradiation, or treatment with a mutagenic substance, such as N-methyl-N'-nitro-N-nitrosoguanidine, etc., and then selecting those that exhibit autotrophy, analogous resistance, or a metabolic regulatory mutation and that also has an ability to produce a target substance. Furthermore, a bacterium that produces a target substance can also be obtained by improving the activity of a target substance biosynthesis enzyme by gene recombination.
From here on, examples of method for providing an ability to produce a target substance, and the microorganisms in which an ability to produce a target substance is provided will be explained.
[00121] Examples of method for providing or improving an ability to produce a target substance in cultivation include, for example, a method of modifying a microorganism such that the expression of a gene encoding an enzyme involved in the biosynthesis of a target substance is better. For example, examples of enzyme involved in L-glutamic acid biosynthesis include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltBD), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase, pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (ene), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk-3) -phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), methyl citrate synthase (prpC), among others. Gene names are described in parentheses after enzyme names (the same format applies to the descriptions below).
[00122] The expression of the aforementioned genes can be improved by the method described for improving the activities of the enzymes of the aforementioned NXA pathway.
[00123] Examples of microorganisms modified in such a way that the expression of the citrate synthase gene, pyruvate dehydrogenase gene and/or glutamate dehydrogenase gene among the aforementioned enzyme genes are better, include the microorganisms described in WO00/18935 , European patent application submitted to the public domain 1010755, among others.
[00124] Furthermore, modification to provide the ability to produce L-glutamic acid can also be accomplished by reducing or eliminating the activity of an enzyme that catalyzes a reaction, which is branched from the L-glutamic acid biosynthetic pathway , and produces a compound other than L-glutamic acid. Examples of enzymes that catalyze a reaction, which branches from the L-glutamic acid biosynthetic pathway, and produce a compound other than L-glutamic acid include 2-oxoketoglutarate dehydrogenase, succinate dehydrogenase, isocitrate lyase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, acetyl-CoA hydrase (international patent publication WO2006/057450), among others.
[00125] In order to reduce or eliminate the activity of a target enzyme, a mutation may be introduced into an enzyme gene in a genome by a usual mutagenesis method, or genetic recombination technique, such that the intracellular activity of the enzyme is reduced or eliminated. Such introduction of a mutation can be achieved, for example, using genetic recombination to eliminate the gene encoding the enzyme in the genome or modify an expression control sequence such as a promoter or the Shine-Dalgarno (SD) sequence. It can also be achieved by introducing an amino acid substitution mutation (sense mutation), a stop codon (sense mutation), or a frameshift mutation by adding or deleting one or two nucleotides in the enzyme-coding regions of the genome , or which partially or completely eliminate the gene (J. Biol. Chem., 272:8611-8617, 1997). Enzyme activity can also be decreased or eliminated by constructing a gene encoding a mutant enzyme, whose coding region is totally or partially deleted, and replacing it with a normal gene in a genome by homologous recombination or the like, or by introducing a transposon or factor IS in the gene.
[00126] For example, in order to introduce a mutation that decreases or eliminates the activities of the aforementioned enzymes by genetic recombination, the following methods are used. A mutant gene is prepared by modifying a partial sequence of a target gene in such a way that it does not encode an enzyme that can function normally, and then a bacterium belonging to the Enterobacteriaceae family can be transformed with a DNA containing the mutant gene to carry out the recombination of a gene in the genome with the mutant gene, to replace the mutant gene with the target gene in the genome. Examples of such gene replacement that use homologous recombination include methods of using a linear DNA such as the method termed Red-directed integration (Datsenko, KA, and Wanner, BL, 2000, Proc. Natl. Acad. Sci. USA, 97 :66406645), and the method using Red-directed integration in combination with an excessive system, directed from phage À (Cho, EH, Gumport, RI, Gardner, JF, 2002, J. Bacteriol., 184:5200 -5203, reference to WO2005/010175, Russian patent application 2006134574), a method of using a plasmid containing a temperature-sensitive origin of replication (US patent 6,303,383, Japanese patent published in the public domain 05-007491), among others . Furthermore, such site-specific mutagenesis based on gene replacement, using homologous recombination, can also be performed using a plasmid that is not capable of replicating in a host.
[00127] Furthermore, the ability to produce L-glutamic acid in coryneform bacteria can also be achieved by a method of amplifying the yggB gene (NCgl 1221;NP_600492. Low conductance reports. [gi:19552490], WO2006/070944) , and a method of introducing a mutant yggB gene, in which a mutation is introduced into the coding region.
[00128] Examples of methods to improve the ability to produce L-glutamic acid include the introduction of genes encoding D-xylulose-5-phosphate phosphoketolase and/or fructose-6-phosphate phosphoketolase (these are collectively called phosphoketolase). Examples of microorganisms that exhibited better phosphoketolase activity include the following microorganisms (WO2006/016705): Brevibacterium lactofermentum ATCC 13869ΔsucA (pVK9-xfp) Brevibacterium lactofermentum ATCC 13869ΔsucA (pVK9-PS2_xpkA)
[00129] The ability to produce L-glutamic acid can also be provided by improving 6-phosphogluconate dehydratase activity, 2-keto-3-deoxy-6-phosphogluconate aldolase activity, or both activities. Examples of microorganism whose 6-phosphogluconate dehydratase activity and 2-keto-3-deoxy-6-phosphogluconate aldolase activity are higher include the microorganism disclosed in Japanese patent filed in the public domain 2003-274988. Furthermore, the ability to produce L-glutamic acid can also be provided by amplifying the yhfK and ybjL genes, which are genes associated with L-glutamic acid secretion (WO2005/085419, WO2008/133161).
[00130] Just as a microorganism that produces L-glutamic acid is used in the present invention, a microorganism with an ability to accumulate L-glutamic acid in a liquid medium in an amount that exceeds the saturation concentration of L-glutamic acid when grown in an acidic condition (hereinafter also referred to as an L-glutamic acid accumulation capacity in an acidic condition), can be used. For example, obtaining a strain in which resistance to L-glutamic acid in a low pH environment is provided, according to the method described in European patent application submitted to the public domain 1078989, the ability to accumulate L-glutamic acid in an amount exceeding the saturation concentration can be supplied.
[00131] As well as methods of providing or improving the ability to produce L-glutamic acid, methods of providing resistance to an organic acid analogue, respiratory inhibitor or the like, and methods of providing sensitivity to an inhibitor of L-glutamic acid can also be mentioned. cell wall synthesis. Examples include, for example, the method of providing resistance to monofluoroacetic acid (Japanese Patent Public Domain 50-113209), the method of providing adenine resistance or thymine resistance (Japanese Patent Public Domain 57065198), the method of attenuating urease (Japanese patent submitted to the public domain 52-038088), the method of providing resistance to malonic acid (Japanese patent submitted to the public domain 52-038088), the method of providing resistance to benzopyrones or naphthoquinones (Japanese patent submitted to the public domain 56-1889), the method of providing resistance to HOQNO (Japanese patent submitted to the public domain 56140895), the method of providing resistance to a-ketomalonic acid (Japanese patent submitted to the public domain 57-2689), the method of providing guanidine resistance (Japanese patent submitted to the public domain 56-35981), the method of providing penicillin sensitivity (Japanese patent submitted to the domain Public address 4-88994), among others.
[00132] Specific examples of such resistant bacteria include the following strains:
[00133] Brevibacterium flavum AJ3949 (FERM BP-2632, reference to Japanese patent submitted to the public domain 50-113209)
[00134] Corynebacterium glutamicum AJ11628 (FERM P-5736, reference to Japanese patent submitted to the public domain 57-065198)
[00135] Brevibacterium flavum AJ11355 (FERM P-5007, reference to Japanese patent submitted to the public domain 56-1889)
[00136] Corynebacterium glutamicum AJ11368 (FERM P-5020, reference to Japanese patent submitted to the public domain 56-1889)
[00137] Brevibacterium flavum AJ11217 (FERM P-4318, reference to Japanese patent submitted to the public domain 57-2869)
[00138] Corynebacterium glutamicum AJ11218 (FERM P-4319, reference to Japanese patent submitted to the public domain 57-2869)
[00139] Brevibacterium flavum AJ11564 (FERM BP-5472, reference to Japanese patent submitted to the public domain 56-140895)
[00140] Brevibacterium flavum AJ11439 (FERM BP-5136, reference to Japanese patent submitted to the public domain 56-35981)
[00141] Corynebacterium glutamicum H7684 (FERM BP-3004, reference to Japanese patent submitted to the public domain 04-88994)
[00142] Brevibacterium lactofermentum AJ11426 (FERM P-5123, reference to Japanese patent submitted to the public domain 56-048890)
[00143] Corynebacterium glutamicum AJ11440 (FERM P-5137, reference to Japanese patent submitted to the public domain 56-048890)
[00144] Brevibacterium lactofermentum AJ11796 (FERM P-6402, reference to Japanese patent submitted to the public domain 58-158192)
[00145] Preferred examples of microorganisms with the ability to produce L-glutamine are bacteria whose glutamate dehydrogenase activity is improved, bacteria whose glutamine synthetase (glnA) activity is improved, and bacteria whose glutaminase gene is disrupted (patent applications European Union submitted to the public domain 1229121 and 1424398). Enhancement of glutamine synthetase activity can also be achieved by disruption of glutamine adenylyltransferase (glnE) or disruption of PII control protein (glnB). Furthermore, a strain belonging to the genus Escherichia and with a mutant glutamine synthetase, in which the tyrosine residue at position 397 is replaced by another amino acid residue, can also be exemplified as a preferred L-glutamine producing bacterium (application published patent US 2003/0148474).
[00146] Other methods of providing or improving the ability to produce L-glutamic acid are the method of providing resistance to 6-diazo-5-oxo-norleucine (Japanese Patent Public Domain 3-232497), the method of providing resistance to purine analogue and resistance to methionine sulfoxide (Japanese patent submitted to the public domain 61-202694), the method of providing resistance to a-ketomalonic (Japanese patent submitted to the public domain 56-151495), among others. Specific examples of coryneform bacteria capable of producing L-glutamic acid include the following strains.
[00147] Brevibacterium flavum AJ11573 (FERM P-5492, Japanese Patent Public Domain 56-161495)
[00148] Brevibacterium flavum AJ11576 (FERM BP-10381, Japanese Patent Public Domain 56-151495)
[00149] Brevibacterium flavum AJ12212 (FERM P-8123, Japanese Patent Public Domain 61-202694)
[00150] Examples of microorganisms capable of producing L-proline include, for example, bacteria with Y-glutamyl kinase that is insensitive to inhibition of response by L-proline, and bacteria whose L-proline decomposition system is attenuated. The method of modifying bacteria using a DNA encoding Y-glutamyl kinase insensitive to response inhibition by L-proline is disclosed in Dandekar, A.M., Uratsu S.L., J. Bacteriol., 170, 12:5943-5 (1988). Furthermore, examples of the method for obtaining a bacterium whose L-proline decomposition system is attenuated include, for example, a method of introducing a mutation into a proline dehydrogenase gene to reduce enzymatic activity. Examples of bacteria capable of producing L-proline include the Escherichia coli NRRL B-12403 strain and the NRRL B-12404 strain (English patent 2075056), the Escherichia coli VKPM B-8012 strain (published patent application US 2002/0058315) , and strains with the mutant plasmid disclosed in German patent 3127361 or with the mutant plasmid disclosed in Bloom FR reference et al. (The 15th Miami Winter Symposium, 1983, p.34).
In addition, preferred L-proline producing microorganisms also include the Escherichia coli 702 (VKPMB-8011) strain, which is a strain resistant to 3,4-dehydroxyproline and azetidine-2-carboxylate, the 702ilvA strain (VKPMB-8012 strain), which is an ilvA-deficient strain of strain 702, the E. coli strains whose protein activity encoded by the b2682, b2683, b1242 or b3434 genes is improved (Japanese Patent Public Domain 2002-300874 ), between others.
[00152] Examples of coryneform bacteria strains that produce L-proline include the strain resistant to DL-3,4-dehydroproline (FERM BP-1219, US patent 4,224,409), strains whose citrate synthetase activity increases by 1, 4 times or more compared to parental strains of these (FERM P5332, FERM P-5333, FERM P-5342, FERMP-5343, Japanese patent 1426823), and the strain in which acetic acid auxotrophy is provided (FERM P-5931 ).
[00153] Examples of microorganism with an ability to produce L-arginine include mutant strains of Escherichia coli with resistance to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamate, AEC (S-(2-aminoethyl)- cysteine), α-methylserine, β-2-thienylalanine, or sulfaguanidine (Japanese Patent Public Domain Reference 56-106598). The Escherichia coli 237 strain, which is a strain that produces L-arginine and carries very active N-acetylglutamate synthase, with a mutation for resistance to response inhibition by L-arginine (Russian patent application 2000117677), is also a bacterium which produces preferred L-arginine. Strain 237 was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) (GNII Genetika) on April 10, 2000 with an accession number of VKPM B-7925, and the original deposit was converted to an international deposit based on the Budapest Treaty on May 18, 2001. The Escherichia coli 382, which is a derivative of the 237 strain and is a L-arginine-producing strain with better ability to assimilate acetic acid (Japanese Patent Public Domain 2002-017342), may also be used. The strain of Escherichia coli 382 was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) on April 10, 2000 under the accession number of VKPM B-7926.
[00154] As well as the microorganism with an ability to produce L-arginine, microorganisms in which the amount of expression of one or more genes encoding a biosynthetic enzyme L-arginine is greater can also be used. Examples of the biosynthetic enzyme L-arginine include one or more enzymes selected from N-acetylglutamate synthetase (argA), N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase ( argD), acetylornithine deacetylase (argE), ornithine carbamoyl transferase (argF), arginine succinic acid synthetase (argG), arginine succinic acid lyase (argH), and carbamoyl phosphate synthase (carAB). It is more preferable to use a mutant N-acetylglutamate synthase (argA) gene encoding the enzyme, in which the amino acid sequence corresponding to positions 15 to 19 of a wild-type enzyme is replaced, and the response inhibition by L-arginine it is hereby canceled (European patent application submitted to the public domain 1170361).
[00155] Although coryneform bacteria that produce L-arginine are not particularly limited, as long as a coryneform bacterium with an ability to produce L-arginine is chosen, examples include wild type strains of coryneform bacteria; coryneform bacteria resistant to certain agents, including sulfa-based drugs, 2-thiazolalanine, α-amino-and-hydroxyvaleric acid, among others; coryneform bacteria that exhibit auxotrophy for L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine or L-tryptophan, in addition to resistance to 2-thiazolalanine (Japanese Patent Public Domain 54-44096); coryneform bacteria resistant to ketomalonic acid, fluoromalonic acid, or monofluoroacetic acid (Japanese Patent Public Domain 57-18989); argininol-resistant coryneform bacteria (Japanese Patent Public Domain 62-24075); coryneform bacteria resistant to X-guanidine (X represents an aliphatic acid derivative or aliphatic chain, Japanese patent submitted to the public domain 2-186995), among others.
[00156] A coryneform bacteria with the ability to produce L-arginine can be cultivated as a mutant strain resistant to 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine among others; a mutant strain resistant to arginine hydroxamate and 2-thiouracil; mutant strain resistant to arginine hydroxamate and 6-azauracil (Japanese Patent Public Domain 49-126819); strain resistant to a histidine analogue or tryptophan analogue (Japanese Patent Public Domain 52-114092); auxotrophic mutant strain for at least one of methionine, histidine, threonine, proline, isoleucine, lysine, adenine, guanine and uracil (or precursor of uracil) (Japanese Patent Public Domain 52-99289); mutant strain resistant to arginine hydroxamate (Japanese patent publication No. 51-6754); mutant strain auxotrophic for succinic acid or resistant to a nucleic acid base analogue (Japanese Patent Public Domain 58-9692); mutant strain deficient in the capacity of arginine decomposition, resistant to an arginine and canavanine antagonist and auxotrophic for lysine (Japanese Patent Public Domain 52-8729); mutant strain resistant to arginine, arginine hydroxamate, homoarginine, D-arginine and canavanine, or resistant to arginine hydroxamate and 6-azauracil (Japanese Patent Public Domain 53-143288); mutant strain resistant to canavanin (Japanese Patent Public Domain 53-3586) or similar.
[00157] Specific examples of coryneform bacteria capable of producing L-arginine include the following strains.
[00158] Brevibacterium flavum AJ11169 (FERM P-4161)
[00159] Brevibacterium lactofermentum AJ12092 (FERM P-7273)
[00160] Brevibacterium flavum AJ11336 (FERM P-4939)
[00161] Brevibacterium flavum AJ11345 (FERM P-4948)
[00162] Brevibacterium lactofermentum AJ12430 (FERM BP-2228)
[00163] In addition, a strain deficient in ArgR, which is an arginine repressor (published patent application US 2002/0045223), and a strain in which glutamine synthetase activity is higher (published patent application) can also be used US 2005/0014236).
[00164] L-citrulline and L-ornithine share common biosynthetic pathways to L-arginine, and the ability to produce L-citrulline and L-ornithine can be improved by increasing the enzymatic activities of N-acetylglutamate synthase (argA), N- acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), and acetylornithine deacetylase (argE) (WO2006/35831).
Like the bacterium that produces Y-aminobutyric acid (GABA), a strain whose glutamate decarboxylase activity is better (Microb. Cell Fact., 2010, Nov. 12;9:85; Amino Acids, 2010 Nov., 39(5):1107-16; published patent application US 2010/0324258) can be used.
[00166] As well as the bacterium that produces putrescine, a strain whose 4-hydroxybutyrate reductase, succinyl-CoA reductase (which forms aldehyde), and 4-hydroxybutyrate dehydrogenase are better (WO2011/047101), and a strain whose Y-aminobutyraldehyde dehydrogenase is better (FEBS Lett., 1 Aug 2005, 579 (19):4107-12) can be used. 2. Method to produce target substance
[00167] By cultivating a bacterium like this, in the manner described above, in a medium containing xylose as a carbon source to produce and accumulate a target substance in the medium, and collecting the target substance from the medium, the target substance can be produced.
[00168] As with the medium used for culture, common media containing a carbon source, nitrogen source and mineral salts, as well as trace organic nutrients such as amino acids and vitamins, can be used as required. Either a synthetic medium or a natural medium can be used.
[00169] As well as the carbon source, as long as xylose is present, other carbon sources, for example, sugars such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, arabinose, hydrolyzed starches and molasses can be used. Furthermore, organic acids such as acetic acid and citric acid, and alcohols such as ethanol can also be used alone or in combination with other carbon sources.
[00170] Although the mixing ratio of xylose and other carbon sources is not particularly limited, the ratio of xylose: other carbon source (weight ratio) is preferably 1:0.1 to 100, more preferably 1:0, 1 to 10, more preferably 1:0.1 to 5, even more preferably 1:1 to 5, further preferred 1:1 to 3.
[00171] The concentration of the carbon source in the medium is not particularly limited, as long as a suitable concentration to produce a target substance is chosen. Meanwhile, the concentration of carbon source in the medium is preferably about 0.1 to 50% w/v, more preferably about 0.5 to 40% w/v, particularly preferably about 1 to 30%.
[00172] Xylose or a mixture of xylose and a hexose, such as glucose, can be obtained from a biomass supply source that is not fully utilized. Such pentoses and hexoses can be released from the biomass by hydrolysis with steam and/or concentrated acid, hydrolysis with dilute acid, hydrolysis with an enzyme such as cellulase, or an alkaline treatment. When the substrate is a cellulose-like material, the cellulose is hydrolyzed to saccharides simultaneously or successively, and the saccharides can be used to produce the target substance. Since hemicellulose is generally easier to hydrolyze to saccharides compared to cellulose, it is preferable to hydrolyze a cellulose-like material beforehand, separate the pentoses, and then hydrolyze the cellulose by a treatment with steam, acid, alkali, cellulase, or a combination of these to produce hexoses.
The xylose contained in the medium used in the present invention can also be provided by converting each of the hexoses to xylose (D-xylose), using a microorganism prepared to present a pathway to convert glucose, galactose or arabinose to xylose.
[00174] As well as the source of nitrogen, ammonia, urea, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitric acid salts, among others can be used . As well as trace organic nutrients, amino acids, vitamins, fatty acids, nucleic acids, those containing the above substances such as peptone, casamino acid, yeast extract, soy protein breakdown product, among others, can be used. When an auxotrophic mutant strain that requires an amino acid or the like for growth is used, it is preferable to supplement with the required nutrient. As well as mineral salts, phosphoric acid salts, magnesium salts, calcium salts, iron salts, manganese salts, among others, can be used.
[00175] The culture is preferably carried out under aerobic conditions, while the fermentation temperature is controlled to be 20 to 45°C, and the pH to be 3 to 9. For pH adjustment, an inorganic or organic acidic or alkaline substance, gas ammonia, among others, can be used. A substantial amount of a target substance is accumulated in the culture medium or cells preferably after 10 to 120 hours of culture under such conditions, in the manner described above.
[00176] Furthermore, when the target substance is L-glutamic acid, the culture can be carried out to produce and accumulate L-glutamic acid with precipitation of L-glutamic acid in a medium using, as the medium, a liquid medium adjusted to satisfy a condition in which L-glutamic acid is precipitated. Examples of the condition in which L-glutamic acid is precipitated include, for example, pH from 5.0 to 4.0, preferably 4.5 to 4.0, more preferably 4.3 to 4.0, particularly preferably 4 .0. In order to simultaneously obtain both improved growth in an acidic condition and efficient precipitation of L-glutamic acid, the pH is preferably 5.0 to 4.0, more preferably 4.5 to 4.0, even more preferably 4 .3 to 4.0. Cultivation can be carried out at the aforementioned pH for the entire cultivation period or a part of it.
[00177] The collected target substance may contain microbial cells, media components, moisture, and microorganism by-product metabolites, in addition to the target substance. The purity of the collected target substance is 50% or more, preferably 85% or more, particularly preferably 95% or more (Japanese patent 1214636, US patents 5,431,933, 4,956,471, 4,777,051, 4,946,654, 5,840 .358, 6,238,714, published patent application US 2005/0025878).
[00178] The target substance obtained in the present invention can be collected from the culture medium, after completion of the culture, by a combination of conventionally known methods such as the ion exchange resin method (Nagai, H. et al. , Separation Science and Technology, 39(16), 3691-3710), membrane separation (Japanese Patent Public Domain Nos. 9-164323 and 9-173792), crystallization (WO2008/078448, WO2008/078646), and others methods.
[00179] In addition, when the target substance deposits in the medium, it can be collected by centrifugation, filtration or the like. A target substance deposited in the medium and a target substance dissolved in the medium can be isolated together, after the target substance dissolved in the medium is crystallized. Examples
[00180] From here on, the present invention will be explained even more specifically with reference to the examples.
[00181] The media compositions used in the examples that follow are shown below. [LB medium] Bacto tryptone 10 g/L Yeast extract 5 g/L NaCl 5 g/L pH 7.0 [LBGM9]
[00182] The same components of the LB medium and the components of the minimal medium (5 g/L glucose, 2 mM magnesium sulfate, 3 g/L monopotassium phosphate, 0.5 g/L sodium chloride, 1 g/L of ammonium chloride, 6 g/L of disodium phosphate) [MSII-Glucose Medium] Group A Glucose 40 g/L MgSOr7H<) 0.5 g/L Group B (NH4)2SO4 20 g/L KH2PO4 2 g/L NaCl 0.5 g/L Yeast extract 2 g/L CaCVVHO 0.25 g/L FeS()/7|l2() 20 mg/L MnSO4mH2O 20 mg/L Trace elements* 4 mL/L L-Lys 200 mg/L DL-Met 200 mg/L DAP 200 mg/L
The components of groups A and B were separately autoclaved at 120°C for 20 minutes, and then mixed. *Trace elements CaCk^HO 0.66 g/L ZnSO/7|2() 0.18 g/L CuSO4^5H2O 0.16 g/L MnSO44H2O 0.15 g/L CoCMHO 0.18 g/L H3BO3 0.10 g/L Na2MoO4 0.30 g/L [MSII-Xylose Medium]
[00184] Same components as MSII-Glucose medium, with the exception that glucose (40 g/L) is replaced by xylose (40 g/L) [MSII-GX Medium]
[00185] Same components as MSII-Glucose medium, with the exception that glucose (40 g/L) is replaced by a mixture of glucose (20 g/L) and xylose (20 g/L) [MSII medium -SX]
[00186] Same components as MSII-Glucose medium, with the exception that glucose (40 g/L) is replaced by a mixture of sucrose (20 g/L) and xylose (20 g/L). [Synthetic medium E1] Group A NH4Cl 20 mM MgSOrVI I2O 2 mM Na2HPO4 40 mM KH2PO4 30 mM CaCl2 0.01 mM FeSO47H2O 0.01 mM MnSO44 in 5H2O 0.01 mM Citrate 5 mM free pH Filter sterilization Group B-1 Source carbon 50 (or 100) mM Filter sterilization Group B-2 Thiamine HCl 1 mM This component was added to the group B-1 component after filtration sterilization (0.22 µm). Group C MES-NaOH (pH 6.8) 50 mM Filter sterilization (0.22 µm)
[00187] The solutions containing the components of groups A to C in concentrations 5 times higher were prepared as stock solutions. [CM-Dex Medium] Polypeptone 10 g/L Yeast Extract 10 g/L Glucose 5 g/L KH2PO4 1 g/L Urea 3 g/L MgSO47H2O 0.4 g/L FeSO47H2O 0.01 g/L MnSO4^ 5H2O 0.01 g/L Soy filtered (hydrolyzed soy) 1.2 g/L (TN) pH 7.5 adjusted with KOH [Glc Medium] Glucose 80 g/L (NH4)2SO4 30 g/L KH2PO4 1 g/ L MgSO47H2O 0.4 g/L FeSO47H2O 0.01 g/L MnSO4^5H2O 0.01 g/L Vitamin B1 200 μg/L Biotin 60 μg/L Filtered soybean (hydrolyzed soybean) 0.48 g/L (TN) pH 8.0 adjusted with KOH [Xyl medium (restricted biotin)] Same components as Glc medium, with the exception that glucose (80 g/L) is replaced by xylose (80 g/L), and biotin is not is contained. [MS Medium] Group A Glucose or Xylose 40 g/L Glucose and Xylose (1:1) 40 g/L MgSOrVIbO 1 g/L Group B (NH4)2SO4 20 g/L KH2PO4 1 g/L Yeast extract 2 g /L FeS()/7|l;() 10 mg/L MnSO4mH2O 10 mg/L
[00188] The components of groups A and B were autoclaved separately at 120°C for 20 minutes, and then mixed, and 50 g/L of calcium carbonate was added, according to the Japanese pharmacopoeia. Example 1: Introduction of the NXA pathway in Pantoea ananatis (1) Construction of plasmid pTWV228Ptac_ccrNXA for introduction of the NXA pathway
[00189] As a known bacterium for which the NXA pathway has been reported, C. crescentus is known (Stephens, C. et al., J. Bacteriol., 189(5):181-2185, 2007). To obtain the genes encoding the C. crescentu NXA pathway enzymes, the following methods were employed.
[00190] The genome of C. crescentus has a size of about 4 Mb, in which the five genes form an operon structure (Journal of Bacteriology, 189:2181-2185, 2007). The genome was extracted from a published genome strain of C. crescentus (CB-15 (ATCC 19089), available as ATCC), and gene cloning and expression vector construction for the genes were tested.
[00191] Expression vectors were constructed using the Clontech IN-Fusion cloning kit.
[00192] The following four types of DNA fragments were amplified by PCR using chromosomal DNA from C. crescentus CB-15 (ATCC 19089) for the following i), ii) and iii), and pMW119 for the following iv) as the molds. The oligonucleotide primers used for PCR are indicated in parentheses. i) tac promoter sequence (hereinafter referred to as "Ptac", PtwvPtacf: SEQ ID NO:1, 0823Ptacr: SEQ ID NO:2) ii) Fragment containing xylX, ccrxylA, ccrxylB and xylC (Ptac0823f: SEQ ID NO: 3, 0819r: SEQ ID NO: 4) iii) xylD and region downstream thereof, of about 120 bp (0819f: SEQ ID NO: 5, 219cc0819r: SEQ ID NO: 6) iv) pMW119/SmaI (219f: SEQ ID NO: 7, 219r: SEQ ID NO: 8)
Next, by PCR using the purified PCR products from i) and ii) as a template, as well as PtwvPtacf and 0819r as the oligonucleotide primers, a Ptac_xylXccrAccrBC fragment consisting of the preceding PCR products ligated together was amplified. The fusion reaction was performed with these three of Ptac_xylXccrAccrBC and the PCR products from iii) and iv) obtained using the Clontech IN-Fusion cloning kit, the E. coli JM109 strain was transformed with the reaction product, and the Target plasmid pMW119 Ptac_ccrNXA was obtained from a transformant.
[00194] Next, using pMW119 Ptac_ccrNXA as the template, as well as PtwvPtacf and 219CC0819r as the oligonucleotide primers, the ccrNXA operon containing Ptac was amplified. The fusion reaction was carried out with the amplified product obtained and pTWV228 is digested with SmaI, the E. coli JM109 strain was transformed with the reaction product, and the target plasmid pTWV228Ptac_ccrNXA was obtained from a transformant. (2) Construction of pUT-MuKm plasmid containing pUT399 bearing kanamycin resistant Mini-A
[00195] pUT399 is a plasmid with the origin of replication of R6K and mob region required for conjugative transfer, and is not replicable in a strain that does not have the pir gene (available from Biomedal, reference to R. Simon., et al. ., BIO/TECHNOLOGY Nov. 1983, 784-791; US patent 7,090,998).
[00196] pCE1134 (Japanese patent submitted to public domain 2-109985) is a plasmid containing MudII1734, and carries a Km resistance gene and lacXYZ gene in the Mini-Mu unit. By the method described below, a DNA fragment lacking the lacXYZ region was prepared from the Mini-Mu unit of pCE1134, and cloned into pUT399.
[00197] From PCR using pCE1134 as the template, as well as oligonucleotide primers attL-F (SEQ ID NO: 9) and nptII-R (SEQ ID NO: 10), a fragment containing the MuCts gene repressor was obtained MuAB, which encodes the left end and transposase, and the Km resistance gene. Furthermore, using pCE1134 as the template, as well as the oligonucleotide primers attR-F (SEQ ID NO: 11) and attR-R (SEQ ID NO: : 12), a fragment containing the right end was obtained in a similar way. Crossover PCR was performed using these 2 fragments as the template, as well as the oligonucleotide primers attL-F and attR-R, and the obtained fragment of about 2.3 kb was introduced into pUT399 at the SmaI site. In this way, plasmid pUT-MuKm was obtained.
[00198] Since the Mini-Mu unit constructed in the manner described above has the Km resistance gene, and the 8-base NotI site it recognizes as a cloning site in the transposition unit, several genes can be cloned into it. . (3) Replacement of drug resistance gene from pTWV228Ptac_ccrNXA
[00199] The ampicillin resistance gene of pTWV228Ptac_ccrNXA was replaced by the kanamycin resistance gene, by the ÀRed method.
[00200] Using pUT_MuKm as the template, as well as the oligonucleotide primers Ap-Km-fw (SEQ ID NO: 13) and Ap-Km-rv (SEQ ID NO: 14), a sequence containing the kanamycin resistance gene ( ntpII fragment) was amplified.
[00201] This PCR was performed using PrimeSTAR HS polymerase (Takara Bio), according to the protocol attached to this enzyme.
A helper plasmid RSF_Red_TER (published patent application US 2009/0286290A1, WO2008/075483) was introduced into E. coli JM109 with pTWV228Ptac_ccrNXA, and cells were cultured in 50 ml of LB medium (containing 1 mM IPTG, 100 mg /L ampicillin, and 25 mg/L chloramphenicol) at 37°C until the OD660 value becomes 0.4.
[00203] The aforementioned RSF_Red_TER is a helper plasmid to introduce À-dependent integration (Red-directed integration, ÀRed method), and can induce the expression of gam, bet and exos genes of with the lacI gene. This plasmid also contains the levansucrase (sacB) gene, and can eliminate a plasmid from a cell with this gene, in a medium containing sucrose. In addition, this plasmid also contains the chloramphenicol resistance gene.
[00204] Cells cultured in the manner described above were collected, washed twice with a 10% glycerol solution by centrifugation, and suspended in 1 mL of a 10% glycerol solution. Next, the cells were transformed with the ntpII fragment previously obtained by electroporation, and the transformants were subjected to selection on LB agar medium containing 40 mg/L of kanamycin. The transformants obtained were inoculated on LB agar medium (containing 1 mM IPTG, 10% sucrose, and 40 mg/L kanamycin), and cultured overnight at 37°C to obtain a single clone. It was confirmed that the obtained transformant could not grow on the medium with LB agar containing 100 mg/L ampicillin, and thereby it was confirmed that the ampicillin resistance gene of pTWV228Ptac_ccrNXA was replaced by the kanamycin resistance gene. The plasmid obtained was named pTWVPtac_ccrNXA_Km. (4) Construction of plasmid containing xylD
Construction was performed using the Clontech IN-Fusion cloning kit.
[00206] First, by PCR using a plasmid containing pUC18, where each gene was cloned as the template, as well as xylD_IFS_5742-10-5 (SEQ ID NO: 15) and xylD_IFS_5742-10-6 (SEQ ID NO: 16) as the oligonucleotide primers, a DNA fragment containing xylD was amplified. Specifically, it was cloned into pUC18, whose SfiI site was removed by the method described below.
[00207] By PCR using the genomic DNA of the C. crescentus strain CB-15 as the template, CC0819-01F_4691-88-7 (SEQ ID NO: 17) and CC0819-01R_5659-9-1 (SEQ ID NO: 18) , as well as CC0819-02F_5659-9-2 (SEQ ID NO:19) and CC0819-02R_4691-88-10 (SEQ ID NO:20) as the oligonucleotide primers, the 1130 bp and 653 bp fragments were amplified, respectively. Next, SmaI-digested pUC18 and the two aforementioned amplified fragments were assembled by the in vitro assembly method (Nature Methods, 6(5), 343-345, 2009) to obtain pUC18-xylD, in which the SfiI site was removed, and the xylD gene was inserted.
Separately, pSTV28-Ptac-Ttrp was digested with SmaI in a conventional manner. The fusion reaction was performed with the xylD gene DNA fragment and the vector DNA fragment, the E. coli strain JM109 was transformed with the reaction product, and the target plasmid pSTVPtac_xylD_Ttrp was obtained from a transformant.
[00209] The pSTV28-Ptac-Ttrp was constructed as follows.
[00210] A DNA fragment (PtacTtrp), with the tac promoter (with the sequence of SEQ ID NO: 32) and the trp terminator sequence, was synthesized and ligated between the KpnI-BamHI sites of the pMW219 vector to obtain pMW219- Ptac-Ttrp. The same amounts of pSTV28 and pMW219-Ptac-Ttrp digested with both KpnI and BamHI were mixed and ligated, JM109 was transformed with the ligation product, and a plasmid was extracted from a colony that showed resistance to Cm. It is confirmed that the plasmids obtained showed bands of about 400 bp and 3 kbp (exactly 389 bp and 2994 bp), which were expected as a result of double digestion with KpnI and BamHI, and thus pSTV28-Ptac-Ttrp was obtained . (4) Production of L-glutamic acid with Pantoea ananatis introduced in the NXA pathway
The P. ananatis NA1 strain was transformed with pTWVPtac_ccrNXA_Km by electroporation (U.S. patent reference 6,682,912). Regarding the strain introduced with pTWVPtac_ccrNXA_Km, a plate medium comprising LBGM9 supplemented with kanamycin, at a final concentration of 40 mg/L, was used.
The cells of the P. ananatis NA1 strain and the transforming strain, grown overnight at 34°C in the LBGM9 plate, were each scraped in an amount corresponding to 1/6 of the plate, inoculated into 5 ml of MSII-Xylose or MSII-GX medium contained in a large test tube, and grown at 34°C and 120 rpm for 48 hours, and residual saccharide, accumulated amounts of L-glutamic acid (Glu) and xylonic acid were measures. The results are shown in Tables 2 and 3. Table 2 (Glu production in MSII-GX medium)
Table 3 (Glu production in MSII-Xylose medium)

[00213] When the P. ananatis NA1 strain was cultivated with the mixed carbon source of glucose and xylose (MSII-GX medium), the glutamic acid yield was 25.7% (Table 2). In this case, accumulation of xylonic acid was observed and thus it is suggested that most of the xylose was converted to xylonic acid. It is estimated that xylonic acid accumulation with the P. ananatis NA1 strain was provided by the glucose dehydrogenase activity of P. ananatis.
[00214] On the other hand, when the P. ananatis NA1 strain introduced with pTWVPtac_ccrNXA_Km was cultivated with the mixed carbon source of glucose and xylose (MSII-GX medium), it showed a much higher yield of glutamic acid compared to the parental strain (yield: 69.9%). If it is taken into account that the parental strain produces a lot of glutamic acid from xylose, and it is assumed that the yield of glutamic acid from the glucose of the strain introduced with pTWVPtac_ccrNXA_Km is equivalent to that of the parental strain, the yield of glutamic acid produced from xylose via the NXA pathway can be estimated as about 86%. In fact, when culture was performed with xylose as a single carbon source (MSII-Xylose), the strain introduced with pTWVPtac_ccrNXA_Km produced Glu in a yield of 80% (Table 3). Example 2: Introduction of the NXA pathway in Escherichia coli (1) Expression of the NXA pathway in E. coli
[00215] Using a strain deficient in isocitrate dehydrogenase (Δicd), which is an enzyme of the TCA cycle and produces αKG from isocitric acid, the expression of the NXA pathway was assayed by growth complementation, in a minimal medium containing xylose as a only source of carbon. Since the icd gene deficient strain cannot produce αKG, it cannot grow on a minimal medium containing xylose as a single carbon source, but if the ability to produce αKG from xylose can be transmitted by introducing the NXA pathway , this strain acquires the ability to grow in an environment like this.
[00216] Specifically the JW1122 strain, which is a strain deficient with respect to the icd gene from the Keio Collection (http://cgsc.biology.yale.edu/Person.php ID99553, available from the E. coli Genetic Resource Center at Yale CGSC, The Coli Genetic Stock Center) was used as a bacterial host strain, and by introducing and expressing the NXA pathway in this strain using a plasmid, it was examined whether the NXA pathway could function in E. coli as well.
[00217] In table 4, the constructed plasmids and the results of growth complementation in the deficient strain with respect to the icd gene are shown. It was confirmed that the strain introduced with a plasmid pMW119 Ptac_ccrNXA (prepared in example 1), containing the NXA pathway operon (xylX, ccrxylA, ccrxylB, xylC, xylD) and the tac promoter in combination, can grow in the M9 minimal medium ( plate) (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)) containing xylose as a single carbon source.
[00218] A similar study was also performed by liquid culture. Growth (OD) on M9 minimal medium and E1 synthetic medium, containing xylose or αKG as a single carbon source, was evaluated over time using a 36-sample culture apparatus. The results are shown in Figure 1. As with the plate culture, the strain introduced in the NXA pathway grew favorably on M9 or E1 medium containing xylose as a single carbon source, whereas the vector control strain did not grow on a medium. like this. It is considered that these results were obtained as a result of the strain growing assimilating xylose through the NXA pathway, that is, the NXA pathway derived from C. crescentus also worked in E. coli. Table 4

(2) Expression of the NXA pathway in the E. coli strain that produces L-glutamic acid
[00219] As well as the E. coli strain that produces L-glutamic acid, MG1655ΔsucA (published U.S. patent application 2005/0106688), which is a strain deficient with respect to αKGDH, was used. pMW119 Ptac_ccrNXA or pMW119 was introduced as a control in the previous strain to obtain MG1655ΔsucA/pMW119Ptac_ccrNXA and MG1655ΔsucA/pMW119. These strains were each grown with culture and flask in MS culture medium containing glucose (40 g/L), xylose (40 g/L), or glucose and xylose (20 g/L each) as the carbon source. Cultivation was carried out for 24 hours on a medium containing only glucose as the carbon source, and for 48 hours on other media. The results are shown in Figure 2. The numerals 325, 425 and 513 appended to the strain names shown in the figure are the clone numbers.
[00220] When the mixed system of glucose and xylose was used as the carbon source, while the control strain (MG1655ΔsucA/pMW119) accumulated 15 to 16 g/L of L-glutamic acid, the strain expressing the ccrNXA operon (MG1655ΔsucA) /pMW119Ptac_ccrNXA) showed L-glutamic acid accumulation at about 12 g/L and thus tended to show reduced L-glutamic acid accumulation and yield. Likewise, when xylose was used as a single carbon source, the same result was obtained. As well as by-products, organic acids and xylonic acid were analyzed. As a result, it is observed that mainly acetic acid and xylonic acid have accumulated. (3) Analysis of the rate limiting point of the NXA pathway in E. coli
[00221] Since xylonic acid, which is an intermediate of the NXA pathway, was detected in the culture supernatant of the E. coli bacteria that expresses the ccrNXA operon and is a producer of L-glutamic acid, in the manner described above, it appears that most likely part of the incorporated xylose was assimilated through the NXA pathway. In addition, the following issues were evaluated. i) Although xylose incorporated into cells can be assimilated both by the system that assimilates xylose, characteristic of E. coli, and by the NXA pathway, a certain amount of xylose can be used by the system characteristic of E. coli, due to the difference in activity or substrate specificity of the first enzyme of the characteristic E. coli system, xylose isomerase (XylA), and of the first enzyme of the NXA pathway, xylose dehydrogenase (XDH) and thus the metabolic flow rate that passes through the NXA pathway can become smaller. ii) It may be a rate limiting point in the NXA pathway, or an unknown shortcut pathway and therefore αKG cannot be produced.
[00222] It is considered that the problem of i) can be solved by increasing the amounts of enzymes in the NXA pathway by changing the expression vector of the NXA operon, from a vector of low copy number type (pMW119) to a vector of the average copy number type (pTWV228), and thereby increasing the amount of substrate consumption in the ccrNXA pathway.
[00223] It is also considered that the problem of ii) can be overcome by improving a strain with cultivation, based on the analysis of the rate limitation point and its results.
[00224] Based on the above consideration, the following was performed: a) construction of a strain from a strain deficient in the xylose assimilation pathway specific for E. coli, (ΔsucAΔxylA), as a host, in which the operon ccrNXA is expressed and thus carbon flux is forced through the NXA pathway, and evaluation of it by cultivation, b) construction of an expression vector of the ccrNXA operon using an average copy number vector, construction of a strain using an expression vector like this, and evaluation of these by cultivation, and c) rate limiting point analysis.
[00225] Deleting the xylA gene that assimilates E. coli specific xylose from MG1655ΔsucA, according to the /-Red method, and using the oligonucleotide primers xylA-H1P1-5742-5-1 (SEQ ID NO: 21) and xylA-H2P2-5742-5-2 (SEQ ID NO:22), strain MG1655ΔsucAΔxylA was obtained. pMW119Ptac_ccrNXA was introduced into this strain to obtain a strain expressing the xylA deficient ccrNXA operon.
[00226] The results of the culture for production of L-glutamic acid, performed by cultivating the strain that expresses the ccrNXA operon deficient in xylA, in the same way described in section (3) above, are shown in figure 3. In figure 3, “ ccrNXA" represents pMW119Ptac_ccrNXA, and the numbers below represent clone numbers.
[00227] While the vector control strain of the ΔsucAΔxylA strain cannot assimilate xylose, and can form cells and produce L-glutamic acid from glucose alone, the strain expressing the ccrNXA operon has been shown to consume xylose and produce L acid -glutamic, which was considered to be the derived form of xylose. However, it is observed that the amount of L-glutamic acid accumulation was lower than that obtained with the model strain (ΔsucA strain) and accumulated xylonic acid, which is a metabolic intermediate of the ccrNXA pathway. From these results, it is suggested that the metabolic flux of the ccrNXA pathway may be insufficient. Furthermore, since the underproduction of αKG was not observed, it is considered that the supply of NADPH required for the expression of GDH activity did not cause any problem at this stage.
[00228] Next, an expression vector of the ccrNXA operon was constructed using an average copy number vector. The ccrNXA operon containing the tac promoter region was amplified using pMW119 Ptac_ccrNXA as the template, as well as PtwvPtacf (SEQ ID NO: 1) and 0819r (SEQ ID NO: 4) as the oligonucleotide primers. pTWV228 was digested with SmaI and used together with the PCR fragment of the ccrNXA operon, containing the tac promoter region, to carry out the fusion reaction, the E. coli JM109 strain was transformed with the reaction product, and the target plasmid pTWVPtac_ccrNXA was obtained from a transformant. This plasmid was introduced into strain MG1655ΔsucA, and the strain obtained was cultured in the same way as described in section (3) above. The results are shown in Figure 4. In the figure, “ΔsucA” represents the MG1655ΔsucA strain, and “/pTWV” and “/v” signify that the strain carried pTWV228. Furthermore, pTWV110 to pTWV119 indicate clone numbers of pTWV228Ptac_ccrNXA.
[00229] When only glucose was used as the carbon source, the strain expressing the ccrNXA operon with average copy number accumulated L-glutamic acid in an amount substantially equivalent to that observed with the control strain. However, when the glucose and xylose mixed culture system was used, the accumulation of L-glutamic acid tended to decrease. Furthermore, the results of sister strains also varied. One of the possible reasons was the elimination of the average copy number expression vector. Furthermore, as in the strains described above, xylonic acid accumulation was observed.
[00230] In order to confirm whether the activities of enzymes of the ccrNXA pathway increased by increasing the number of copies of the NXA operon, the activity of XDH, which is the first enzyme of the NXA pathway, was evaluated. The results are shown in table 5. “7513” and “1110” in the strain names mentioned in table 5 are the clone numbers. Table 5: Results of the evaluation of XDH (xylose dehydrogenase) activity
ND: not detected Note: Relative activity was reported as the relative activity based on the specific activity of ccrNXA7513 determined to be 1.
[00231] The strain introduced with expression vector with medium copy number showed XDH activity about 7 times higher, compared to the strain introduced with expression vector with low copy number. It was not confirmed how xylose was actually distributed in cells from the point of derivation of xylose isomerase (XylA), characteristic of E. coli and XDH, and thus it was also considered that the activity of XDH, which is the first enzyme NXA pathway, may be insufficient. However, since the increase in XDH activity did not provide the effect that improves L-glutamic acid accumulation, and based on xylonic acid accumulation among others, it is considered that any one or more of the NXA pathway enzymes may be rate limiting.
[00232] Therefore, the rate limiting point of the NXA pathway was analyzed. Since xylonic acid accumulated as a metabolic intermediate, it was considered at least that the rate limiting point may exist in the xylonic acid pathway to αKG, not the xylose to xylonic acid pathway. Furthermore, in the structure of the ccrNXA operon, while enzymes from the xylose to xylonic acid pathway are encoded by genes located in the third and fourth positions, enzymes from the xylonic acid pathway to αKG are encoded by the genes located in the first, second and fifth positions positions. The activity of XDH, whose gene is located at the third position in the operon, was detected in vitro, and so it was considered that if the enzyme activity encoded by the gene located at the fifth position in the operon (XylD) could be detected additionally, this it can function as circumstantial evidence of transcription and translation of the complete NXA operon. Therefore, it is estimated that one of the three reactions from xylonic acid to αKG constituted a rate limiting point, and the following experiments were performed.
Plasmids pSTVPtac_xylD_Ttrp, pSTVPtac_xylX_Ttrp, and pSTVPtac_ccrxylA_Ttrp expressing the genes xylD, xylX and ccrxylA, respectively, were prepared as follows.
pSTV28-Ptac-xylX-Ttrp was prepared by preparing a xylX fragment by PCR using pUC18-xylX, which is a plasmid prepared by cloning xylX, from which the SfiI site was removed in pUC18 as the template, as well as xylX-IFS-5742-10-1 (SEQ ID NO: 38) and xylX-IFA-5742-10-2 (SEQ ID NO: 39) as the oligonucleotide primers, and cloning the plasmid obtained into pSTV28-Ptac- Ttrp digested with SmaI by the fusion cloning method.
[00235] pSTV28-Ptac-ccrxylA-Ttrp was prepared by preparing a ccrxylA fragment by PCR using pUC18-ccrxylA, which is a plasmid prepared by cloning ccrxylA, from which the SfiI site was removed in pUC18 as the template as well as xylA_IFS_5742-10-3 (SEQ ID NO: 40) and xylA_IFA_5742-10-4 (SEQ ID NO: 41) as the oligonucleotide primers, and cloning the obtained plasmid into pSTV28-Ptac-Ttrp digested with SmaI by the cloning method in Fusion.
[00236] pSTV28-Ptac-xylD-Ttrp was prepared by preparing an xylD fragment by PCR using pUC18-xylD, which is a plasmid prepared by cloning xylD, from which the SfiI site was removed in pUC18 as the template as well as xylD_IFS_5742-10-5 (SEQ ID NO: 42) and xylD_IFA_5742-10-6 (SEQ ID NO: 43) as the oligonucleotide primers, and cloning the plasmid obtained into pSTV28-Ptac-Ttrp digested with SmaI by the cloning method in Fusion.
The plasmids pUC18-xylX, pUC18-ccrxylA and pUC18-xylD mentioned above were prepared in the manner described below, respectively.
[00238] By PCR using the genomic DNA of the C. crescentus strain CB-15 as the template, CC0823-01F_4691-87-1 (SEQ ID NO: 44) and CC0823-01R_4691-87-2 (SEQ ID NO: 45 ), as well as CC0823-02F_4691-87-3 (SEQ ID NO: 46) and CC0823-02R_4691-87-4 (SEQ ID NO: 47) as the oligonucleotide primers, the 900 bp and 280 bp fragments were amplified, respectively . Next, pUC18 digested with SmaI, and two of the aforementioned amplified fragments were assembled by the in vitro assembly method (Nature Methods, 6(5), 343-345, 2009) to obtain pUC18-xylX, in which the SfiI site was removed, and the xylX gene was inserted.
[00239] By PCR using the genomic DNA of the C. crescentus strain CB-15 as the template, CC0822-01F_4691-87-5 (SEQ ID NO: 48) and CC0822-01R_5659-8-7 (SEQ ID NO: 49 ), CC0822-02F_5659-8-8 (SEQ ID NO:50) and CC0822-02R_5659-8-9 (SEQ ID NO:51), CC0822-03F_5659-8-10 (SEQ ID NO:52) and CC0822-03R_5659 -8-11 (SEQ ID NO: 53), CC0822-04F_5659-8-12 (SEQ ID NO: 54) and CC0822-04R_5659-8-13 (SEQ ID NO: 55), as well as CC0822-05F_5659-8- 14 (SEQ ID NO: 56) and CC0822-05R_4691-87-14 (SEQ ID NO: 57) as the oligonucleotide primers, five fragments, 11v02 (175 bp), 12v02 (325 bp), 13v02 (260 bp), 14v02 (193 bp) and 15v02 (544 bp) were amplified, respectively. Next, two of the 11v02 and 12v02 fragments were ligated by crossover PCR using these two fragments as the template, as well as CC0822-01F_4691-87-5 and CC0822-02R_5659-8-9 as the oligonucleotide primers. Similarly, two of the 13v02 and 14v02 fragments were ligated by crossover PCR using these two fragments as the template, as well as CC0822-03F_5659-8-12 and CC0822-04R_5659-8-13 as the oligonucleotide primers. These two fragments and the aforementioned 15v02 fragment were joined by the in vitro assembly method (Nature Methods, 6(5), 343-345, 2009). The ligated fragment obtained was amplified by PCR using it as the template, as well as CC0822-01F_4691-87-5 and CC0822-05R_4691-87-14 as the oligonucleotide primers. Next, pUC18 digested with SmaI, and the aforementioned ligated fragment was assembled by the in vitro assembly method (Nature Methods, 6(5), 343-345, 2009) to obtain pUC18-ccrxylA, in which the SfiI site was removed , and the ccrxylA gene was inserted.
[00240] By PCR using the genomic DNA of the C. crescentus strain CB-15 as the template, CC0819-01F_4691-88-7 (SEQ ID NO: 17) and CC0819-01R_5659-9-1 (SEQ ID NO: 18 ), as well as CC0819-02F_5659-9-2 (SEQ ID NO: 19) and CC0819-02R_4691-88-10 (SEQ ID NO: 20) as the oligonucleotide primers, the 1130 bp and 653 bp fragments were amplified, respectively . Next, pUC18 digested with SmaI, and two of the aforementioned amplified fragments were assembled by the in vitro assembly method (Nature Methods, 6(5), 343-345, 2009) to obtain pUC18-xylD, in which the SfiI site was removed, and the xylD gene was inserted.
[00241] Crude enzyme extracts were prepared from the strain that expresses the ccrNXA operon (MG1655ΔsucA/pTWV228Ptac_ccrNXA), and from the strains that carry each of the plasmids that express one of the aforementioned genes xylD, xylX, and ccrxylA, respectively ( MG1655ΔsucA/pSTVPtac_xylD_Ttrp, MG1655ΔsucA/pSTVPtac_xylX_Ttrp, and MG1655ΔsucA/pSTVPtac_ccrxylA_Ttrp), and then each of the crude enzyme extracts from the strain that carries only one of the xyl and xyl genes that expresses the xyl and xylD vectors were added to the xyl, or vector expressing genes. crude enzyme extract from the strain expressing the ccrNXA operon, and the activity to produce αKG from the xylonic acid of each mixture was measured. The results are shown in Table 6. In Table 6, “1,110” appended to the strain name is the clone number. Table 6: Activity measurement results to produce αKG from xylonic acid
Note: The relative activity is indicated as the relative activity based on the specific activity of the ccrNXA1110 and pSTV28-Ptac-Ttrp mixed system determined as 1.
[00242] With the system in which crude enzyme extract from the strain that expresses only the xylD gene was added, the increased activity that produces αKG was observed. From this result, it is suggested that the xylonate dehydratase (XylD) encoded by the xylD gene constituted a rate limiting point of the NXA pathway, built in E. coli by heterogeneous expression.
[00243] Since the metabolic flux of the complete pathway may be better by further increasing the expression of the xylD gene in the strain expressing the ccrNXA operon, in the manner suggested by the measurement of enzymatic activity, a strain with better expression of the xylD gene was constructed by introducing a xylD gene expression vector in the strain expressing the ccrNXA operon, and was evaluated by cultivation for L-glutamic acid production using glucose and xylose as the carbon source. As well as the strain expressing the ccrNXA operon, MG1655ΔsucA/pMW119Ptac_ccrNXA and MG1655ΔsucA/pTWV228Ptac_ccrNXA were used.
[00244] Cultivation was carried out in the same manner as that described in section (3) mentioned above.
[00245] The results are shown in table 7. The strain that improves xylD gene expression showed much better L-glutamic acid accumulation and yield. The accumulation of L-glutamic acid was 23 to 25 g/L, as opposed to 15 to 16 g/L of the control strain, and the yield based on consumed saccharide reached 57 to 60%, as opposed to 37 to 40% of the control strain. Xylonic acid, which is a metabolic intermediate, was not observed in the strain with better expression of the xylD gene. On the other hand, the effect of improving the expression of the xylD gene was observed only in the expression strain that carries an expression vector of the ccrNXA operon of the medium copy number type, and the effect was not observed in the expression strain that carries the vector of the low number of copies type. From these results, it is considered that removing the rate limiting point of this pathway, increasing the activity of the complete NXA pathway by increasing the vector copy number, and further improving the expression of the xylD gene provided the improvement. in the amount of production of L-glutamic acid. Furthermore, when the activity to produce αKG from xylonic acid of the strain with the best expression of the xylD gene was evaluated, it increased about 10-fold compared to that observed before the improvement (Table 8). The numbers “1,110”, “17” and “19” attached to the strain names, mentioned in tables 7 and 8, are the clone numbers. Table 7: Result of the evaluation of the strain expressing ccrNXA + xylD operon by L-Glu production culture
Table 8: Activity improvement results to produce αKG from xylonic acid
Note: Results for MG1655ΔsucA/pTWVccrNXA1110 are values obtained from experiments using different lots. ND: Not Detected Note: Relative activity is indicated as relative activity based on the specific activity of ccrNXA1110 determined as 1. Example 3: Introduction of NXA pathway in Corynebacterium glutamicum (1) Construction of plasmid pVK9Peftu_ccrNXA for introduction of NXA pathway
[00246] A plasmid with a sequence containing the promoter sequence of the Tu elongation factor gene (EF-Tu), tuf (WO2008/114721, SEQ ID NO: 33, hereinafter referred to as "Peftu") and xylD, linked downstream of the promoter sequence, was constructed using the HD Clontech In-Fusion cloning kit (Clontech). First, PCR was performed using chromosomal DNA from the C. glutamicum ATCC 13869 strain as the template, as well as oligonucleotide primers Peftu(Pst) (SEQ ID NO: 58) and Peftu_Rv (SEQ ID NO: 59) to obtain a fragment containing the Peftu sequence. This PCR was performed using PrimeSTAR HS polymerase, according to the protocol attached to this enzyme.
[00247] Furthermore, PCR was performed using pTWV228Ptac_ccrNXA as the template, as well as oligonucleotide primers Peftu_xylXABCD_fw (SEQ ID NO: 60) and Peftu_xylXABCD_rv (SEQ ID NO: 61), to obtain a fragment containing the sequence xylXABCD of C. crescentus . This PCR was performed using PrimeSTAR GXL polymerase, according to the protocol attached to this enzyme.
[00248] Next, the Peftu fragment and the fragment containing xylXABCD obtained above were mixed with pVK9, treated with PstI and BamHI, and used to carry out the fusion reaction according to the Clontech In-fusion HD cloning kit protocol. pVK9 is a transport vector obtained by pHSG299 with blunt end (Takara Bio) at the AvaII site, and inserting an autonomously replicable region in coryneform bacteria contained in pHK4 (Japanese patent submitted to the public domain 05-007491), which was excised with BamHI and KpnI and presented a blunt end (Japanese patent submitted to the public domain 200797573, published patent application US 2005/0196846). E. coli JM109 was transformed into the fusion reaction mixture. The transformants were subjected to selection on an agar medium, which comprises LB medium supplemented with kanamycin at a final concentration of 50 mg/L. The target plasmid pVK9Peftu_ccrNXA was obtained from a obtained transformant. (2) L-glutamic acid production by Corynebacterium glutamicum introduced via the NXA pathway
[00249] The C. glutamicum ATCC 13869 strain was transformed with the previously mentioned pVK9Peftu_ccrNXA by the electrical pulse method (Japanese patent submitted to the public domain 2-207791). A strain introduced with pVK9Peftu_ccrNXA was selected on an agar medium, which comprises CM-Dex medium supplemented with kanamycin at a final concentration of 25 mg/L. In addition, the C. glutamicum ATCC13869 strain transformed with pVK9 was also selected in a similar way to a control strain.
[00250] The ability to assimilate xylose and the ability to produce L-glutamic acid of the obtained transformants were verified by performing cultivation using a Sakaguchi flask. Cells from each transformant strain, grown at 31.5°C for 24 hours on CM-Dex agar medium supplemented with kanamycin at a final concentration of 25 mg/L, were scraped in an amount corresponding to 1/6 of the plate , and inoculated in 20 mL of Glc medium contained in a Sakaguchi flask, 1 g of calcium carbonate previously sterilized with hot air was added, and the culture with shaking was carried out at 31.5°C and 120 rpm for 24 hours. The culture medium obtained in a volume of 1 ml was inoculated into 20 ml of Xyl medium (with restricted biotin) contained in a Sakaguchi flask, 1 g of calcium carbonate previously sterilized with hot air was added, and the culture with shaking was performed at 31.5°C and 120 rpm for 73 hours. The results are shown in table 9.
[00251] The C. glutamicum ATCC 13869 strain introduced with pVK9 grew a lot in the Xyl medium, and also did not show the production of L-glutamic acid. On the other hand, the C. glutamicum ATCC 13869 strain introduced with pVK9Peftu_ccrNXA grew in Xyl medium and accumulated L-glutamic acid. From these results, it is observed that the introduction of the NXA pathway in coryneform bacteria improved the ability to assimilate xylose, and a strain like this produced L-glutamic acid from xylose. Table 9
(3) Construction of the strain that further improves xylD gene expression
[00252] Since the C. glutamicum ATCC 13869 strain carrying pVK9Peftu_ccrNXA accumulated xylonic acid, it is assumed that the activity of the xylD gene product was insufficient. Therefore, an attempt was made to construct a plasmid for the expression of the NXA pathway, in which the xylD gene was greatly improved by introducing one or more copies of the xylD gene into plasmid pVK9 Peftu_ccrNXA. (4) Construction of plasmid pVS7PmsrA_xylD for xylD gene expression
A plasmid with a sequence comprising the promoter sequence of the msrA gene (peptide methionine sulfoxide reductase A) (hereinafter referred to as "PmsrA"), and the C. crescentus xylD gene linked downstream of the promoter sequence, was constructed using Hd Clontech In-Fusion cloning kit.
[00254] First, PCR was performed using the chromosomal DNA of the ATCC 13869 strain of C. glutamicum as the template, as well as the oligonucleotide primers PmsrA(Pst) (SEQ ID NO: 62) and PmsrAR (SEQ ID NO: 63) , to obtain a fragment containing the PmsrA sequence. In addition, PCR was performed using pTWV228Ptac_ccrNXA as the template, as well as the oligonucleotide primers PmsrA_xylD_fw (SEQ ID NO:64) and Peftu_xylXABCD_rv, to obtain a fragment containing the C. crescentus xylD gene sequence. These PCRs were performed using PrimeSTAR HS polymerase, according to the protocol attached to this enzyme.
[00255] Next, the fragments containing the PmsrA fragment and the xylD gene obtained above were mixed with the pVS7 transport vector (the construction method is shown below), treated with PstI and BamHI, and used to carry out the reaction on fusion according to the HD Clontech In-fusion cloning kit protocol, and then E. coli JM109 was transformed with this reaction mixture. The transformants were subjected to selection on an agar medium, comprising LB medium supplemented with spectinomycin at a final concentration of 25 mg/L. The target plasmid pVS7PmsrA_xylD was obtained from a obtained transformant.
[00256] pVS7 is a plasmid obtained by replacing the chloramphenicol resistance gene of pVC7 (Japanese patent submitted to the public domain 2000-201692, European Patent 1004671) by a spectinomycin resistance gene. The spectinomycin resistance gene can be obtained by preparing a plasmid pDG1726 from the Escherichia coli ECE101E strain, sold by the Bacillus Genetic Stock Center (BGSC), and extracting the plasmid resistance gene as a cassette. By means of PCR using pDG1726 as the template, as well as SpcR-F (SEQ ID NO: 65) and SpcR-R (SEQ ID NO: 66) oligonucleotide primers, the spectinomycin resistance gene was amplified. The obtained gene fragment was mixed with pVC7 treated with SmaI, a ligation reaction was performed according to the ligation mix protocol <Mighty Mix> from Takara Bio, and E. coli JM109 was transformed with this reaction mix. The transformants were subjected to selection on an agar medium, comprising LB medium supplemented with spectinomycin at a final concentration of 25 mg/L. pVC7-spc comprising pVC7 inserted with the spectinomycin resistance gene was obtained from a obtained transformant. In addition, PCR was performed using pVC7-spc as the template, as well as the oligonucleotide primers spc(GTG start)-F (SEQ ID NO: 67) and spc(stop)-R (SEQ ID NO: 68), for amplify the spectinomycin resistance gene.
[00257] Separately, PCR was performed using pVC7 as the template, as well as the oligonucleotide primers Spc-pVC7-Cm-F (SEQ ID NO: 69) and Spc-pVC7-Cm-R (SEQ ID NO: 70), to obtain a DNA fragment comprising pVC7, in which the chloramphenicol resistance gene has been removed. This DNA fragment and the DNA fragment of the spectinomycin resistance gene, obtained previously from pVC7-spc, were mixed and used to perform the fusion reaction according to the Clontech In-fusion HD cloning kit protocol, and E. coli JM109 was transformed with the resulting reaction mixture. The transformants were subjected to selection on an agar medium comprising LB medium supplemented with spectinomycin at a final concentration of 25 mg/L. pVS7 was obtained from a obtained transformant. (5) Construction of plasmid pVK9Peftu_ccrNXA+D with additional enhancement of xylD gene
[00258] The DNA fragment containing the spectinomycin resistance gene (Spc) and PmsrA_xylD was amplified using pVS7PmsrA_xylD as the template, as well as the oligonucleotide primers ME_Spc_fw (SEQ ID NO: 71) and ME_Peftu_xylXABCD_rv) (SEQ ID NO: 72). The obtained DNA fragment was inserted into pVK9Peftu_ccrNXA in vitro, according to the Ez-Tn5TM Custom Transposome construction kit protocol (Epicentre), and the resultant was used to transform E. coli DH5α. Transformants were subjected to selection on an agar medium comprising LB agar medium supplemented with kanamycin and spectinomycin, at a final concentration of 50 mg/L and 25 mg/L, respectively. Plasmids were extracted from the transformants obtained, and a plasmid for which the insertion site of the Spc-PmsrA_xylD sequence was confirmed not to be in the Peftu_ccrNXA sequence, by nucleotide sequence analysis around the Spc-PmsrA_xylD sequence, was determined pVK9Peftu_ccrNXA+D. (6) Production of L-glutamic acid by the strain introduced in the NXA pathway with additional enhancement of the xylD gene
[00259] pVK9Peftu_ccrNXA+D was introduced into the strain C. glutamicum ATCC 13869 by the electric pulse method, and the cells were applied on CMDex agar medium, containing 25 mg/L of kanamycin. The ability to produce L-glutamic acid, of a strain grown after cultivation at 31.5°C, was verified in the same way as that in section (2) previously mentioned. The results are shown in table 10.
[00260] The strain that carries pVK9Peftu_ccrNXA+D accumulated D-xylonic acid in a smaller amount, but accumulated L-glutamic acid in a greater amount compared to the strain that carries pVK9Peftu_ccrNXA. By this result, it was demonstrated that L-glutamic acid can be more efficiently produced from the D-xylose pathway and from the NXA pathway, further improving the xylD gene. Table 10: L-glutamic acid production of the strain introduced in the NXA pathway with additional enhancement of the xylD gene
Example 4 (1) Replacement of NXA pathway genes
[00261] In the aforementioned examples, using the xylX, ccrxylA, ccrxylB, xylC and xylD genes from a known bacterium C. crescentus, for which the NXA pathway has been reported, it is demonstrated that glutamic acid can be produced from the the xylose pathway to the NXA pathway. In this example, the xylD, xylX, and ccrxylA homolog genes are obtained from biological species other than C. crescentus, and it is investigated whether these genes can replace the C. crescentus genes. As well as the gene sources, the biological species described in table 11 were chosen. Gene symbols of genes (GenBank) are also known. The sequence identification numbers of the nucleotide sequences of the genes, and the amino acid sequences encoded by them, used in the sequence listing are shown in Table 12. In Table 12, "original" means naturally occurring gene nucleotide sequence, and “optimized” means nucleotide sequence whose codons are optimized according to codon usage in E. coli.
[00262] In the descriptions below, the enzymes encoded by the homologues of the genes xylD, xylX and xylA can be referred to as XylD, XylX and XylA, respectively. Table 11
Table 12

(2) Construction of plasmids to detect the activities of XylD, XylX and XylA, pTWVPtac_ccrNXA_ΔxylD_Km, pTWVPtac_ccrNXA_ΔxylX_Km and pTWVPtac_ccrNXA_ΔccrxylA_Km
Construction was performed using the Clontech In-Fusion cloning kit.
[00264] First, by PCR using pTWVPtac_ccrNXA_Km as the template, as well as Ptac_xylXABC_F (SEQ ID NO: 111) and Ptac_xylXABC_R (SEQ ID NO: 112) as the oligonucleotide primers, the DNA fragment was amplified, except for xylD. The PCR product was used to perform the fusion reaction, according to the HD Clontech In-fusion cloning kit protocol, the E. coli JM109 strain was transformed with the reaction product, and the target plasmid pTWVPtac_ccrNXA_ΔxylD_Km was obtained a from a transformant.
[00265] In the same manner as described above, pTWVPtac_ccrNXA_ΔxylX_Km was constructed using Ptac_xylABCD_F (SEQ ID NO: 113) and Ptac_xylABCD_R (SEQ ID NO: 114) as the oligonucleotide primers, and pTWVPtac_ccrNXA_xylABCD_F (SEQ ID NO: 113) and Ptac_xylABCD_R (SEQ ID NO: 114) as the oligonucleotide primers, and pTWVPtac_ccrNXA_Ktac_ccr_CDFxyl was constructed using PtBtac_Xc_SEQ ID NO: 115 (SEQ ID NO: 116) as the oligonucleotide primers. (3) Construction of Pantoea ananatis to detect XylD, XylX and XylA activities
[00266] The P. ananatis NA1 strain was transformed with pTWVPtac_ccrNXA_ΔxylD_Km described previously by the electroporation method. The constructed strain was referred to as P. ananatis NA2 ΔxylD. For the cultivation of P. ananatis NA2 ΔxylD, a plate medium comprising LBGM9 was used, in which kanamycin and tetracycline were added at final concentrations of 40 mg/L and 12.5 mg/L, respectively.
[00267] Similarly, the P. ananatis NA1 strain was transformed with pTWVPtac_ccrNXA_ΔxylX_Km or pTWVPtac_ccrNXA_ΔccrxylA_Km to build the P. ananatis NA2 strain ΔxylX and the P. ananatis NA2 Δccrxyl strain. (4) Construction of expression plasmids homologous to xylD, xylX and xylA
The plasmids for the expression of yjhG or yagF, pSTV28-Ptac-yjhG-Ttrp and pSTV28-Ptac-yagF-Ttrp, were prepared as follows.
[00269] pSTV28-Ptac-yjhG-Ttrp was prepared by amplifying a yjhG fragment by PCR that uses genomic DNA from E. coli strain MG1655 as the template, as well as yjhG_F (SEQ ID NO: 117) and yjhG_R (SEQ ID NO : 118) as the oligonucleotide primers, and cloning the amplified fragment into pSTV28-Ptac-Ttrp digested with SmaI, according to the fusion cloning method.
pSTV28-Ptac-yagF-Ttrp was prepared in the same manner as described above using yagF_F (SEQ ID NO: 119) and yagF_R (SEQ ID NO: 120) as the oligonucleotide primers.
[00271] A plasmid for the expression of xylD(Hse), pSTV28-Ptac-xylD(Hse)-Ttrp, was prepared as follows. A DNA fragment with the sequences of the tac promoter, xylD(Hse) and trp terminator (Ptac-xylD(Hse)-Ttrp), was synthesized and ligated to pUC57 vector (obtained from Thermo Fischer Scientific) digested with EcoRV to obtain pUC57- Ptac-xylD(Hse)-Ttrp. When the DNA fragment was synthesized, the codons were optimized so that the fragment was suitable for expression in E. coli. Equal amounts of pSTV28 and pUC57-Ptac-xylD(Hse)-Ttrp, both of which were digested with EcoRI and KpnI, were mixed and the ligation reaction was carried out. Next, JM109 was transformed with the ligation product, and a plasmid was extracted from a colony that exhibited resistance to Cm to obtain pSTV28-Ptac-xylD(Hse)-Ttrp.
[00272] Plasmids for expression of xylD(Amis), xylD(Aor), xylX(Cne), xylX(Zga), xylX(Tco), xylA(Abr), and ycbD, pSTV28-Ptac-xylD(Amis)- Ttrp, pSTV28-Ptac-xylD(Aor)-Ttrp, pSTV28-Ptac-xylX(Cne)-Ttrp, pSTV28-Ptac-xylX(Zga)-Ttrp, pSTV28-Ptac-xylX(Tco)-Ttrp, pSTV28-Ptac xylA(Abr)-Ttrp and pSTV28-Ptac-ycbD-Ttrp, respectively, were also prepared in the same way. Codon optimization was not performed for YcbD.
[00273] A plasmid for the expression of xylD(Atu), pSTV28-Ptac-xylD(Atu)-Ttrp, was prepared as follows. A DNA fragment with the sequences of the tac promoter, xylD(Atu) and trp terminator (Ptac-xylD(Atu)-Ttrp) was synthesized and ligated to the pJET1.2 vector (obtained by Thermo Fischer Scientific), digested with EcoRV, for obtain pJET1.2-Ptac-xylD(Atu)-Ttrp. When the DNA fragment was synthesized, the codons were optimized in such a way that the fragment was suitable for expression in E. coli. Equal amounts of pSTV28 and pJET1.2-Ptac-xylD(Atu)-Ttrp, both of which were digested with EcoRI and KpnI, were mixed and the ligation reaction was carried out. Next, JM109 was transformed with the ligation product, and a plasmid was extracted from a colony exhibiting resistance to Cm to obtain pSTV28-Ptac-xylD(Atu)-Ttrp.
Plasmids for the expression of xylX(Atu) or xylA(Hbo), pSTV28-Ptac-xylX(Atu)-Ttrp and pSTV28-Ptac-xylA(Hbo)-Ttrp were also prepared in the same way.
A plasmid for the expression of xylX(Art), pSTV28-Ptac-xylX(Art)-Ttrp, was prepared as follows. A DNA fragment with the sequences of the tac promoter, xylX(Art) and trp terminator (Ptac-xylX(Art)-Ttrp) was synthesized and ligated to pCC1 vector (obtained from Epicentre), digested with EcoRV, to obtain pCC1-Ptac -xylX(Art)-Ttrp. When the DNA fragment was synthesized, the codons were optimized in such a way that the fragment was suitable for expression in E. coli. Equal amounts of pSTV28 and pCC1-Ptac-xylX(Art)-Ttrp, both of which were digested with EcoRI and KpnI, were mixed and the ligation reaction was carried out. Next, JM109 was transformed with the ligation product, and a plasmid was extracted from a colony exhibiting resistance to Cm to obtain pSTV28-Ptac-xylX(Art)-Ttrp. (5) Detection of XylD homologs activities
The P. ananatis NA2 ΔxylD strain was transformed with pSTV28-Ptac-Ttrp, pSTV28-Ptac-xylD-Ttrp, pSTV28-Ptac-xylD(Atu)-Ttrp, pSTV28-Ptac-xylD(Hse)-Ttrp, pSTV28-Ptac-yjhG-Ttrp, pSTV28-Ptac-yagF-Ttrp, pSTV28-Ptac-xylD(Amis)-Ttrp or pSTV28-Ptac-xylD(Aor)-Ttrp by the electroporation method (reference to US patent 6,682,912) . For the cultivation of the transformants, a plate medium comprising LBGM9 was used, in which kanamycin, tetracycline and chloramphenicol were added at final concentrations of 40 mg/L, 12.5 mg/L and 25 mg/L, respectively.
[00277] The cells of each transformant grown overnight at 34°C in the plate with LBGM9, in which the drugs were added, were scraped in an amount corresponding to 1/6 of the cells on the plate, inoculated into 5 ml of the MSII-SX medium contained in a large test tube, and cultured at 34°C and 120 rpm for 48 hours, and the amount of L-glutamic acid (Glu) accumulated was evaluated. The results are shown in Figure 5.
[00278] While the P. ananatis NA2 ΔxylD strain introduced with pSTV28-Ptac-Ttrp accumulated 6.9 g/L of L-glutamic acid, the other transformants accumulated 11.1 to 23.1 g/L of L-glutamic acid . In the strain introduced with pSTV28-Ptac-Ttrp, L-glutamic acid was largely produced from xylose and, thus, it was considered that L-glutamic acid was produced from xylose through the NXA pathway in other transformants. That is, it has been shown that a homologue of xylD, derived from any biological species other than C. crescentus, can be substituted for xylD. (6) Detection of activities of XylX counterparts
The P. ananatis NA2 ΔxylX strain was transformed with pSTV28-Ptac-Ttrp, pSTV28-Ptac-xylX-Ttrp, pSTV28-Ptac-xylX(Art)-Ttrp, pSTV28-Ptac-xylX(Atu)-Ttrp, pSTV28 -Ptac-xylX(Cne)-Ttrp, pSTV28-Ptac-xylX(Zga)-Ttrp, pSTV28-Ptac-xylX(Tco)-Ttrp or pSTV28-Ptac-xylX(Seal)-Ttrp by the electroporation method. Regarding the cultivation of transformants, a plate medium comprising LBGM9 was used to which kanamycin, tetracycline and chloramphenicol were added at final concentrations of 40 mg/L, 12.5 mg/L and 25 mg/L, respectively.
[00280] The cells of each transformant grown overnight at 34°C in the LBGM9 plate, in which the drugs were added, were scraped in an amount corresponding to 1/6 of the cells on the plate, inoculated into 5 ml of the MSII-SX medium contained in a large test tube, and cultured at 34°C and 120 rpm for 48 hours, and the amount of L-glutamic acid (Glu) accumulated was measured. The results are shown in figure 6.
[00281] While the P. ananatis NA2 ΔxylX strain introduced with pSTV28-Ptac-Ttrp accumulated 8.7 g/L of L-glutamic acid, the other transformants accumulated 20.5 to 27.8 g/L of L-glutamic acid . In the strain introduced with pSTV28-Ptac-Ttrp, L-glutamic acid was largely produced from xylose and, thus, it was considered that L-glutamic acid was produced from xylose through the NXA pathway in other transformants. That is, it has been shown that an analog of xylX, derived from any biological species other than C. crescentus, can be substituted for xylX. (7) Detection of activities of XylA counterparts
The P. ananatis NA2 strain ΔccrxylA was transformed with pSTV28-Ptac-Ttrp, pSTV28-Ptac-ccrxylA-Ttrp, pSTV28-Ptac-ycbD-Ttrp, pSTV28-Ptac-xylA(Hbo)-TV28-Pta or pta xylA(Abr)-Ttrp by the electroporation method. Regarding the cultivation of transformants, a plate medium comprising LBGM9 was used, in which kanamycin, tetracycline and chloramphenicol were added at final concentrations of 40 mg/L, 12.5 mg/L and 25 mg/L, respectively.
[00283] The cells of each transformant grown overnight at 34°C in the LBGM9 plate, in which the drugs were added, were scraped in an amount corresponding to 1/6 of the cells on the plate, inoculated into 5 ml of the MSII-SX medium contained in a large test tube, and grown at 34°C and 120 rpm for 48 hours, and the amount of L-glutamic acid (Glu) that had accumulated was measured. The results are shown in Figure 7.
[00284] While the P. ananatis NA2 ΔccrxylA strain introduced with pSTV28-Ptac-Ttrp accumulated 1.0 g/L of L-glutamic acid, the other transformants accumulated 18.1 to 30.7 g/L of L-glutamic acid . In the strain introduced with pSTV28-Ptac-Ttrp, L-glutamic acid was largely produced from xylose and, thus, it was considered that L-glutamic acid was produced from xylose through the NXA pathway in other transformants. That is, it has been shown that a homologue of ccrxylA, derived from any biological species other than C. crescentus, can be substituted for xylA. Industrial Applicability
[00285] According to the present invention, a target substance can be efficiently produced by fermentation using xylose as a raw material.

权利要求:
Claims (7)
[0001]
1. Method for producing a target substance comprising cultivating a bacterium with an ability to produce the target substance, in a medium containing xylose as a carbon source, to produce and accumulate the target substance in the medium, and collect the target substance from the medium, characterized by the fact that: the target substance is 2-ketoglutaric acid or a derivative thereof, the xylose dehydrogenase activity and the xylonolactonase activity were transmitted or increased in bacteria by the introduction of the xylB gene that encodes xylose dehydrogenase and the xylC gene which encodes xylonolactonase in forms that can be expressed in bacteria such that the bacteria has an ability to produce xylonic acid from xylose, xylonate dehydratase activity, 2-keto-3-deoxyxylonate dehydratase activity, and 2-semialdehyde activity ketoglutaric dehydrogenase were provided or improved in bacteria by the introduction of the xylD, yjhG or yagF gene encoding xylonate dehydratase , xylX gene that encodes 2-keto-3-deoxyxylonate dehydratase and the xylA gene that encodes 2-ketoglutaric semialdehyde dehydrogenase in forms capable of expression in the bacterium, the bacterium is a bacterium belonging to the genus Pantoea or to the genus Corynebacterium, and the 2-ketoglutaric acid derivative is a substance selected from the group consisting of L-glutamic acid, L-glutamine, L-arginine, L-citrulline, L-ornithine, L-proline, putrescine and y-aminobutyric acid.
[0002]
2. Method according to claim 1, characterized in that the xylB gene and the xylC gene are each derived from a microorganism belonging to the genus Caulobacter.
[0003]
3. Method according to claim 1 or 2, characterized in that the genes xy1D, yjhG or yagF, gene xylX and gene xy1A are each derived from a microorganism belonging to the genus Caulobacter, Escherichia, Agrobacterium, Herbaspirillum, Actinoplanes, Cupriavidus, Pseudomonas, Zobellia, Thermobacillus, Arthrobacter, Azospirillum, Halomonas, Bacillus or Aspergillus.
[0004]
4. Method according to any one of claims 1 to 3, characterized in that the bacterium has been further modified in such a way that the activity of 2-ketoglutarate dehydrogenase is reduced.
[0005]
5. Method according to any one of claims 1 to 4, characterized in that the bacterium has been further modified in such a way that the succinate dehydrogenase activity is reduced.
[0006]
6. Method according to any one of claims 1 to 5, characterized in that the bacterium is Pantoea ananatis.
[0007]
7. Method according to any one of claims 1 to 5, characterized in that the bacterium is Corynebacterium glutamicum.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-06-30| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-03-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161558685P| true| 2011-11-11|2011-11-11|

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US61/558685|2011-11-11|

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JP2011247031|2011-11-11|

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JP2011-247031|2011-11-11|

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PCT/JP2012/078725|WO2013069634A1|2011-11-11|2012-11-06|Method for producing target substance by fermentation|

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