JP2015228804A - Microorganisms capable of producing 2,3-butanediol and methods for producing 2,3-butanediol using the same, and methods for producing 1,3-butadiene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide microorganisms for efficient and safe production of 2,3-butanediol, where decrease in the microbial growth rate is prevented and byproduct is formed less.SOLUTION: Provided is a microorganism having 2,3-butanediol production ability, modified to decrease the activity of: a) lactate dehydrogenase; b) one of or both of acetate kinase and phosphotransacetylase; and c) CoA transferase, in comparison with the not-modified strain.

Description

  The present invention relates to a novel microorganism having the ability to produce 2,3-butanediol and a method for producing 2,3-butanediol using the same. The present invention also relates to a method for producing 1,3-butadiene using 2,3-butanediol obtained by the production method as a raw material.

2,3-butanediol is a straight-chain hydrocarbon having 4 carbon atoms, and hydroxyl groups are bonded to the 2nd and 3rd positions, respectively, and three types of (R, R), (S, S), and (meso) isomers are used. Optical isomers exist. 2,3-butanediol is a raw material for 1,3-butadiene, which is useful as a raw material for synthetic rubber, and methyl ethyl ketone, which is used as a solvent, by dehydration reaction. In addition, printing ink, fragrance, plasticizer, It is expected to be used for foods, pharmaceuticals, softeners, etc.
In addition, due to factors such as depletion of petroleum resources and environmental impact, attention is now focused on fermentation production using microorganisms. Known microorganisms that naturally fermentatively produce 2,3-butanediol include Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, and the like (for example, Non-Patent Document 1).

  The history of studying the natural fermentation production of 2,3-butanediol is old, and its productivity is relatively high. For example, in the above-mentioned Klebsiella pneumoniae, results of accumulation amount 150 g / L and productivity 4.21 g / L / h have been reported (Non-patent Document 2). On the other hand, these microorganisms are causative bacteria such as pneumonia and enteritis, are designated as BSL2, and have a drawback that they are not suitable for culturing on a large scale for industrial use.

  For this reason, attempts have been made to search for 2,3-butanediol mass-producing bacteria classified into GRAS or BSL1, which can be cultured safely, or to eliminate toxicity by modifying the strain. In addition, attempts have been made to produce 2,3-butanediol by genetic recombination techniques using commonly used Escherichia coli and the like. However, none of the techniques provides a safe microorganism that exhibits productivity comparable to that of natural bacteria.

In addition, for microorganisms used in the production of 2,3-butanediol, the gene involved in the 2,3-butanediol production pathway is enhanced by gene recombination technology, or the gene involved in the byproduct production pathway is destroyed. A technique for improving productivity can also be a useful technique.
For example, Non-Patent Document 3 shows that by using Bacillus licheniformis in which the lactate dehydrogenase gene is disrupted, the by-product of lactic acid is suppressed and the productivity of 2,3-butanediol is improved.

In Patent Document 1, Escherichia coli in which a lactate dehydrogenase gene, a phosphoacetyltransferase gene, an acetate kinase gene, and a pyruvate oxidase gene are disrupted is used to suppress by-production of lactic acid and acetic acid, and 2,3- It has been shown to improve the productivity of butanediol.
Furthermore, Non-Patent Document 4 shows that by using Saccharomyces cerevisiae in which the pyruvate decarboxylase gene is disrupted, ethanol by-product is suppressed and 2,3-butanediol productivity is improved. .

International Publication WO2013 / 076144 Pamphlet

Biotechnol Adv. , 2011, May-Jun 29 (3), p351-64 Appl Microbiol Biotechnol. 2009, Feb 82 (1), p49-57. Biotechnol Bioeng. , 2012, Jul 109 (7), p1610-21 Bioresource Technol. , 2013, Oct 146, p274-81

However, although the method described in Non-Patent Document 3 reduces the by-product of lactic acid, ethanol, formic acid, acetic acid, succinic acid and the like are still by-produced, and there is room for improvement in the production of 2,3-butanediol. was there.
Moreover, although the method of patent document 1 is carrying out the genetic modification which reduces the byproduct of lactic acid and an acetic acid, and is producing 2, 3- butanediol, the accumulation amount is 0.257 g / L at the maximum. The results were low, and there was room for improvement in terms of further reduction of by-products and production of 2,3-butanediol.
Furthermore, the method described in Non-Patent Document 4 has a problem that the growth rate decreases due to the destruction of the pyruvate decarboxylase gene. For this reason, mutant strains whose growth has been recovered have been obtained. However, since this method requires a great deal of labor, a rational modification of microorganisms has been desired.
An object of the present invention is to reduce the production of by-products while suppressing a decrease in the growth rate of microorganisms, and to efficiently and safely produce 2,3-butanediol, and 2,3 using the microorganisms -To provide a method for producing butanediol and 1,3-butadiene.

  As a result of intensive studies to solve the above problems, the present inventors have found that any one of lactate dehydrogenase activity, acetate kinase activity and phosphotransacetylase activity in a microorganism used for production of 2,3-butanediol or Both, as well as reducing CoA transferase activity, while suppressing the decrease in the growth rate of microorganisms, we found that by-products such as lactic acid and acetic acid were reduced and the productivity of 2,3-butanediol was improved, The present invention has been completed.

That is, the said subject is achieved by the following structures.
[1] A microorganism having the ability to produce 2,3-butanediol and modified so that the activities of the following a), b), and c) are reduced as compared with an unmodified strain.
a) lactate dehydrogenase activity b) acetate kinase activity and / or phosphotransacetylase activity c) CoA transferase activity [2] modified to reduce pyruvate oxidase activity compared to unmodified strain, The microorganism according to [1].
[3] The microorganism according to [1] or [2], which has been modified so that pyruvate carboxylase activity is reduced as compared with an unmodified strain.
[4] Further, at least one enzyme activity selected from the group consisting of acetolactate synthase activity, acetolactate decarboxylase activity, 2,3-butanediol dehydrogenase activity, and diacetylreductase activity is enhanced as compared with an unmodified strain The microorganism according to any one of [1] to [3], which has been modified as described above.
[5] The microorganism according to any one of [1] to [4], further modified so that the xylose isomerase activity is enhanced as compared with the unmodified strain.
[6] The microorganism according to any one of [1] to [5], further modified so that the xylulokinase activity is enhanced as compared with the unmodified strain.
[7] The microorganism is a coryneform bacterium, Escherichia coli, Bacillus genus bacteria, Anaerobiospirillum genus bacteria, Actinobacillus genus bacteria, Mannemia genus bacteria, Basfia genus Basf The microorganism according to any one of [1] to [6], which is at least one selected from the group consisting of a bacterium, a genus Zymomonas, a bacterium belonging to the genus Zymobacter, a filamentous fungus, and a yeast.
[8] A method for producing 2,3-butanediol, wherein the microorganism according to any one of [1] to [7] or a processed product thereof is allowed to act on an organic raw material in an aqueous medium.
[9] The method for producing 2,3-butanediol according to [8], wherein the organic raw material contains one or more selected from the group consisting of glucose, sucrose, and xylose.
[10] A step of producing 2,3-butanediol by the method described in [8] or [9], and conversion to 1,3-butadiene using the 2,3-butanediol obtained in the step as a raw material The manufacturing method of 1, 3- butadiene including a process.

According to the present invention, it is possible to provide a microorganism in which the production of by-products is reduced and the productivity of 2,3-butanediol is improved without decreasing the growth rate.
By using such a microorganism of the present invention, 2,3-butanediol can be produced efficiently and safely.

It is a figure which shows the metabolic pathway regarding 2, 3- butanediol production | generation and its by-product production | generation. It is a figure which shows the construction procedure of pC3.14F-AlsSD. It is a figure which shows the construction procedure of pTZ4F-BdhA.

Hereinafter, embodiments of the present invention will be described in detail.
<Microorganism of the present invention>
The microorganism of the present invention is a microorganism having an ability to produce 2,3-butanediol, and has been modified so that the activities of the following a), b), and c) are reduced as compared with the unmodified strain. It is a microorganism.
a) Lactate dehydrogenase activity b) Acetate kinase activity and / or phosphotransacetylase activity c) CoA transferase activity

In the present invention, “2,3-butanediol-producing ability” means that when a microorganism is cultured in a medium, the microorganism can produce and accumulate 2,3-butanediol in the medium.
2,3-butanediol is a straight-chain hydrocarbon having 4 carbon atoms, and hydroxyl groups are bonded to the 2nd and 3rd positions, respectively, and three types of (R, R), (S, S), and (meso) isomers are used. Optical isomers exist. In the present invention, 2,3-butanediol produced by a microorganism may be any isomer of these.

Here, the microorganism having the ability to produce 2,3-butanediol may be a microorganism originally having the ability to produce 2,3-butanediol or imparted the ability to produce 2,3-butanediol by breeding. It may be a thing.
Examples of means for imparting the ability to produce 2,3-butanediol by breeding include mutation treatment and gene recombination treatment. Known methods such as enhancement of enzyme gene expression in the biosynthesis pathway of 2,3-butanediol Can be adopted. Examples thereof include modifications that impart or enhance acetolactate synthase activity, acetolactate decarboxylase activity, 2,3-butanediol dehydrogenase activity, and diacetylreductase activity as described below.

The microorganism used in the present invention is not particularly limited as long as it is a microorganism capable of producing 2,3-butanediol, but is not limited to coryneform bacteria, Escherichia coli, Bacillus bacteria, Anaerobiospirillum bacteria, Actino. A microorganism selected from the group consisting of bacteria belonging to the genus Actinobacillus, bacteria belonging to the genus Mannheimia, bacteria belonging to the genus Basfia, bacteria belonging to the genus Zymomonas, bacteria belonging to the genus Zymomobacter, filamentous fungi, and yeasts Preferably there is.
Among these, at least one selected from the group consisting of coryneform bacteria, Escherichia coli, Bacillus genus bacteria, Actinobacillus genus bacteria, filamentous fungi, and yeasts is preferable, and coryneform bacteria, Bacillus ( Bacillus) bacteria and yeasts, particularly preferably coryneform bacteria.

  The coryneform bacterium is not particularly limited as long as it is classified as such, and examples include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Arthrobacter, and the like. Preferred examples include those belonging to the genus Corynebacterium and Brevibacterium, and more preferred are Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes ( Bacteria classified as Brevibacterium lactofermentum or Brevibacterium lactofermentum A.

  Particularly preferred specific examples of coryneform bacteria that can be used in the present invention include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium Ammonia Genes ATCC 6872, Corynebacterium glutamicum ATCC 31831, Brevibacterium lactofermentum ATCC 13869 and the like. Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl W, Ehrmann M, Ludwig W, Schleiffer KH, Int J Systact Bacteriol., 1991, Vol. 41). p255-260), in the present invention, the Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 are respectively Corynebacterium glutamicum MJ-233 and MJ-233 AB-41. The same stock as the stock.

  The above-mentioned Brevibacterium flavum MJ-233 was founded on April 28, 1975 at the Institute of Microbial Technology of the Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (〒305-8856 Japan) No. 1 in Higashi 1-chome, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan, with deposit number FERM P-3068, transferred to an international deposit based on the Budapest Treaty on May 1, 1981, under the deposit number of FERM BP-1497 It has been deposited.

Examples of E. coli that can be used in the present invention include Escherichia coli.
Examples of bacteria belonging to the genus Bacillus that can be used in the present invention include Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and the like.
Examples of the genus Anaerobiospirillum that can be used in the present invention include Anaerobiospirillum succiniciproducens.

Examples of bacteria belonging to the genus Actinobacillus that can be used in the present invention include Actinobacillus succinogenes.
Examples of the bacterium belonging to the genus Mannheimia that can be used in the present invention include basin succinici product (Mannheimia succiniciproducens) and the like.

Examples of the bacterium belonging to the genus Basfia that can be used in the present invention include Basfia succiniciproducens.
Examples of the genus Zymomonas that can be used in the present invention include Zymomonas mobilis.
Examples of the genus Zymobacter that can be used in the present invention include Zymobacter palmae.

  Examples of filamentous fungi that can be used in the present invention include Aspergillus, Penicillium, and Rhizopus. Among these, Aspergillus niger (Aspergillus oryzae) and the like are Aspergillus niger (Aspergillus oryzae) and the like, and Penicillium chrysogenum (Pencillium chrysogenium and Penicillium crisiumum lysilis) are included in the genus Aspergillus. . In the genus Rhizopus, Rhizopus oryzae and the like can be mentioned.

  Examples of yeasts that can be used in the present invention include Saccharomyces, Shizosaccharomyces, Candida, Pichia, Kluyveromyces (Kluyveromyces), Yarrowia), Zygosaccharomyces.

Examples of the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces uvarum, and Saccharomyces bayanmys.
Examples of the genus Shizosaccharomyces include Schizosaccharomyces pombe.
Examples of the genus Candida include Candida albicans, Candida sonorensis, Candida glabrata, and the like.
Examples of the Pichia genus include Pichia pastoris and Pichia stipitis.

Examples of the genus Kluyveromyces include Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerance (Kluyveromyces thermotolerance (Kluyveromyces thermotolerance). Is mentioned.
Examples of the genus Yarrowia include Yarrowia lipolytica.
Examples of the genus Zygosaccharomyces include Zygosaccharomyces bailii and Zygosaccharomyces rouxii.

  The microorganism is not only a wild strain, but also a mutant strain obtained by a normal mutation treatment such as UV irradiation or NTG treatment, a recombinant strain induced by a genetic technique such as cell fusion or gene recombination. Or a stock of

  As described above, the microorganism of the present invention includes a microorganism having 2,3-butanediol-producing ability, as described later, one or both of lactate dehydrogenase activity, acetate kinase activity and phosphotransacetylase activity, and CoA transferase. It can be obtained by modifying so that the activity is reduced.

  In the present invention, “lactate dehydrogenase (hereinafter also referred to as LDH) activity” refers to an activity (EC: 1.1.1.17) that catalyzes a reaction of reducing pyruvic acid to produce lactic acid. “The LDH activity is decreased” means that the LDH activity is decreased as compared with the unmodified strain. The LDH activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. LDH activity may be completely lost. Decrease in LDH activity is confirmed by measuring LDH activity by a known method such as the method of Kanarek et al. (Kanarek L, Hill RL, J Biol Chem., 1964, Vol. 239, p4202-4206). Can do.

Strains modified to reduce LDH activity use the above-mentioned microorganisms as parent strains, and mutations commonly used for mutation treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid Each can be obtained by selecting a strain treated with an agent and having reduced LDH activity. It can also be achieved by disrupting the ldh gene on the chromosome or modifying expression control sequences such as a promoter or Shine-Dalgarno (SD) sequence. Specifically, in the region of the ldh gene containing the promoter sequence on the chromosome, deletion of a part or all of the region, insertion of the nucleotide sequence to disrupt it, introduction of mutation, to a weaker promoter Substitution etc. are mentioned. A plurality of methods for reducing these LDH activities may be combined.
As a method for producing a strain modified so that LDH activity is reduced, a method by homologous recombination to a chromosome (see JP-A-11-206385, etc.), a method using a sacB gene (Schaffer A, Tach A, Jager W, Kalinowski J, Thierbach G, Puhler A, Gene 1994 Vol. 145 (1), p69-73).

  Hereinafter, a method for disrupting the ldh gene in coryneform bacteria will be described. The ldh gene is obtained, for example, by synthesizing a synthetic oligonucleotide based on the Ldh gene sequence of Corynebacterium glutamicum registered with cg3219 in the sequence of GenBank accession number NC_006958, and the chromosome of Corynebacterium glutamicum. Cloning can be performed by performing a PCR reaction using DNA as a template. Chromosomal DNA is obtained from bacteria that are DNA donors, for example, by the method of Saito and Miura (Saito H, Miura K, Biochim Biophys Acta., 1963, Vol. 72, p619-629, Biotechnology Experiments, Japan Society for Biotechnology, Ed., Pp. 97-98, Bafukan, 1992).

  The ldh gene prepared as described above or a part thereof can be used for gene disruption. However, since the gene used for gene disruption only needs to have homology (identity) to the extent that homologous recombination occurs with the ldh gene on the chromosomal DNA of the bacterium to be disrupted, it is homologous to the ldh gene of coryneform bacteria. A homologous gene having sex can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions (complementary strand of the ldh gene sequence). Here, the stringent conditions are 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, 0.1%, which are the usual washing conditions for Southern hybridization. A condition for hybridization at a salt concentration corresponding to SDS is mentioned.

  Using the gene as described above, for example, a deletion type ldh gene is prepared by deleting a partial sequence of the ldh gene so as not to produce a normal LDH protein, and a coryneform bacterium with a DNA containing the gene. The ldh gene on the chromosome can be destroyed by transformation and recombination between the deletion gene and the gene on the chromosome. Gene disruption by gene replacement using such homologous recombination has already been established, and there are a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383). Or Japanese Patent Laid-Open No. 05-007491). In addition, gene disruption by gene replacement using homologous recombination as described above can be performed using a plasmid that does not have replication ability on the host. As a plasmid that does not have replication ability in coryneform bacteria, A plasmid having replication ability in Escherichia coli is preferable, and examples thereof include pHSG299 (manufactured by Takara Bio Inc.), pHSG399 (manufactured by Takara Bio Inc.), and the like.

  In the present invention, “acetate kinase (hereinafter also referred to as ACK) activity” refers to an activity (EC: 2.7.2.1) that catalyzes a reaction for producing acetic acid from acetyl phosphate and ADP. “The ACK activity is reduced” means that the ACK activity is reduced as compared to the unmodified strain. The ACK activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. Further, the ACK activity may be completely lost. The decrease in ACK activity can be confirmed by measuring the ACK activity by a known method such as the method of Ramponi et al. (Ramponi G, Meth Enzymol., 1975, Vol. 42, p409-426).

  In the present invention, “phosphotransacetylase (hereinafter also referred to as PTA) activity” refers to an activity that catalyzes a reaction of transferring phosphoric acid to acetyl CoA to produce acetyl phosphate (EC: 2.3.1. 8). “PTA activity is reduced” means that PTA activity is reduced as compared to an unmodified strain. The PTA activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. Further, the PTA activity may be completely lost. The decrease in PTA activity can be confirmed by measuring the PTA activity by a known method such as the method of Klotzsch et al. (Klotzsch HR, Meth Enzymol., 1969, Vol. 12, p381-386).

Strains modified to reduce ACK activity and strains modified to reduce PTA activity can be prepared in the same manner as the above-described method for reducing LDH activity. Either one of the ACK activity and the PTA activity may be reduced, but in order to efficiently reduce the by-product of acetic acid, it is more preferable to reduce both activities.
In Corynebacterium glutamicum, as described in Microbiology, 1999, Feb 145 (Pt2), p503-513, both enzymes were encoded by the pta-ack operon, so the pta gene was destroyed. In some cases, the activities of both PTA and ACK enzymes can be reduced.

  In the present invention, “CoA transferase (hereinafter also referred to as CTF) activity” refers to an activity that catalyzes a reaction for producing acetic acid by transferring CoA of acetyl CoA to another substance (EC: 2.8. 3.18). ). “CTF activity is reduced” means that CTF activity is reduced as compared to an unmodified strain. The CTF activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. Moreover, CTF activity may be lose | disappeared completely. The decrease in CTF activity is known in the art, for example, the method of Veit et al. (Veit A, Rittmann D, Georgi T, Youn JW, Eikmanns BJ, Wendish VF, J Biotechnol., 2009, Vol. 140 (1-2). , P75-83) can be confirmed by measuring CTF activity.

Strains modified to reduce CTF activity can be prepared in the same manner as described above for reducing LDH activity.
Moreover, the order is not ask | required about the modification which reduces any one or both of LDH activity, ACK activity, and PTA activity, and CTF activity.

  In microorganisms having the ability to produce 2,3-butanediol, if modification is made to reduce LDH activity, the by-product of lactic acid can be reduced, but the growth rate of the microorganisms is reduced. It was. Therefore, in addition to the modification, if one or both of ACK activity and PTA activity involved in the metabolic pathway for producing acetic acid from acetyl-CoA and CTF activity are modified, acetic acid by-product is produced. In addition to the reduction, the decrease in the growth rate of microorganisms could be greatly suppressed, and the problem when LDH activity was reduced could be solved.

The biosynthetic pathway for 2,3-butanediol and lactic acid is a pathway that consumes reducing power such as NADH. On the other hand, the pathway for biosynthesizing acetic acid from pyruvic acid via acetyl CoA is a pathway for supplying reducing power such as NADH. This is because NADH and the like are produced when pyruvate is converted to acetyl CoA by a pyruvate dehydrogenase complex (hereinafter also referred to as PDHc).
In microorganisms having the ability to produce 2,3-butanediol, when modification is performed to reduce LDH activity, there is a tendency that by-products of lactic acid are reduced and by-products of acetic acid are increased. That is, not only the flux of the path that consumes the reducing power decreases, but also the flux of the path that supplies the reducing power increases. As a result, it was considered that the strain in which only the modification that reduced the LDH activity was performed significantly changed the intracellular redox balance, and the growth rate of the microorganism was reduced.
In a microorganism having the ability to produce 2,3-butanediol, in addition to a modification that reduces LDH activity, a modification that reduces one or both of ACK activity and PTA activity, and CTF activity, Not only lactic acid but also by-product of acetic acid is reduced, and production of 2,3-butanediol is increased. That is, the flux of the pathway that supplies the reducing power is reduced and the flux of the pathway that consumes the reducing power is increased as compared with the case of only modification that reduces the LDH activity. As a result, it was considered that the redox balance in the cells was improved and the decrease in the growth rate of the microorganisms was largely suppressed.

  In addition to the above modification, the microorganism of the present invention is preferably modified so as to reduce pyruvate oxidase activity. “Pyruvate oxidase (hereinafter also referred to as PoxB) activity” refers to an activity (EC: 1.2.5.1) that catalyzes a reaction for producing acetic acid from pyruvic acid and water. “PoxB activity is reduced” means that PoxB activity is reduced as compared to an unmodified strain. The PoxB activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. In addition, PoxB activity may be completely lost. It is confirmed by measuring the PoxB activity by a known method, for example, the method of Chang et al. (Chang YY, Cronan JE JR, J Bacteriol., 1982, Vol. 151, p1279-1289). Can do.

Strains modified to reduce PoxB activity can be prepared in the same manner as described above for reducing LDH activity.
In addition to the modification that reduces LDH activity, ACK activity, and / or PTA activity, and CTF activity, modification that reduces PoxB activity can further reduce the by-product of acetic acid, The productivity of 2,3-butanediol can be further improved.

  The microorganism of the present invention may be modified to reduce pyruvate carboxylase activity in addition to the modifications described above. The “pyruvate carboxylase (hereinafter also referred to as PC) activity” refers to an activity (EC: 6.4.1.1) that catalyzes a reaction in which pyruvate is carboxylated to produce oxaloacetate. “PC activity is reduced” means that PC activity is reduced as compared to an unmodified strain. The PC activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. Further, the PC activity may be completely lost. The decrease in PC activity was confirmed by measuring PC activity by a known method, for example, the method of Fisher et al. (Fisher SH, Masasanik B, J Bacteriol., 1984, Vol. 158 (1), p55-62). can do.

Strains modified to reduce PC activity can be prepared in the same manner as described above for reducing LDH activity.
In addition to modifications that reduce LDH activity, ACK activity and / or PTA activity, and CTF activity, succinic acid, fumaric acid, malic acid, etc. Can be reduced, and the productivity of 2,3-butanediol can be improved.

The microorganism of the present invention may be a microorganism inherently capable of producing 2,3-butanediol or a microorganism imparted with 2,3-butanediol-producing ability by breeding.
Specific examples of modifications that impart 2,3-butanediol-producing ability by breeding are selected from the group consisting of acetolactate synthase activity, acetolactate decarboxylase activity, 2,3-butanediol dehydrogenase activity, and diacetylreductase activity Modifications that impart or enhance at least one enzyme activity.

  Here, “acetolactate synthase (hereinafter also referred to as ALS) activity” refers to an activity (EC: 2.2.1.6) that catalyzes a reaction for producing acetolactate from pyruvic acid. “ALS activity is enhanced” means that ALS activity is higher than that of the unmodified strain. The ALS activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. ALS activity is imparted or enhanced by measuring ALS activity by a known method, for example, Stormer et al. (Stormer FC, J Biol Chem., 1968, Vol. 243 (13), p3735-3739). Can be confirmed.

  Examples of the method of enhancing ALS activity include a method of treating a parent strain with a mutagen, a method of increasing the copy number of the alsS gene encoding ALS, and a method of modifying the alsS gene and the promoter of the alsS gene. Further, a plurality of methods for enhancing these ALS activities may be combined.

Hereinafter, a specific method for producing a strain modified so that ALS activity is imparted or enhanced will be described. In the strain with enhanced ALS activity, the parent strain was treated with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, and the ALS activity was increased. It can be obtained by selecting a strain.
In addition, a strain to which ALS activity has been imparted or enhanced can also be obtained by modification using the alsS gene. Specifically, it can be achieved by increasing the copy number of the alsS gene. The increase in the copy number can be achieved by transforming the gene on the chromosome by a technique such as transformation with a vector containing the alsS gene or homologous recombination. This can be achieved by introducing and making multiple copies on the chromosome.

Furthermore, a strain with enhanced ALS activity can also be achieved by increasing the ALS activity per molecule of protein encoded by the alsS gene by introducing a mutation into the alsS gene on the chromosome or plasmid vector.
Also, a strain with enhanced expression of the alsS gene can be obtained by introducing a mutation into the promoter of the alsS gene on a chromosome or a plasmid vector, or by making the alsS gene highly expressed by replacing it with a stronger promoter. Can also be achieved.

The alsS gene used for imparting or enhancing ALS activity is not particularly limited as long as it encodes a protein having ALS activity, and examples thereof include a gene derived from Bacillus subtilis.
Furthermore, a bacterium other than Bacillus subtilis, or an alsS gene derived from other microorganisms or animals and plants can also be used. For the alsS gene derived from microorganisms or animals and plants, a gene whose base sequence has already been determined or a gene encoding a protein having ALS activity based on homology or the like is isolated from a chromosome of microorganisms, animals or plants, etc. The determined one can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In enhancing ALS activity, a plurality of types of alsS genes derived from different microorganisms or animals and plants may be used.

  An ALS expression vector is provided by inserting the gene encoding ALS isolated as described above into a known expression vector so that the gene can be expressed. By transforming with this expression vector, a strain modified so that ALS activity is imparted or enhanced can be obtained. Alternatively, a strain modified so as to impart or enhance ALS activity can also be obtained by incorporating ALS-encoding DNA into the chromosomal DNA of the host microorganism so as to allow expression, such as by homologous recombination. Note that transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

  When the alsS gene is introduced onto a chromosome or a plasmid, a suitable promoter is incorporated upstream of the gene 5'-side, more preferably a terminator 3'-downstream. The promoter and terminator are not particularly limited as long as the promoter and terminator are known to function in microorganisms used as hosts, and may be the promoter and terminator of the alsS gene itself, or other promoters. And a terminator. Vectors, promoters and terminators that can be used in these various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”.

  Hereinafter, a method for introducing the alsS gene in coryneform bacteria will be described. When a coryneform bacterium is used, a DNA fragment containing the alsS gene can be expressed in the coryneform bacterium by introducing the DNA fragment containing the alsS gene into a plasmid vector containing a gene that controls the replication and replication function of the plasmid in the coryneform bacterium. Recombinant plasmids can be obtained. By transforming a coryneform bacterium such as Corynebacterium glutamicum MJ-233 with the recombinant vector, a coryneform bacterium into which the alsS gene has been introduced can be obtained. Transformation can be performed, for example, by an electric pulse method (Vertes AA, Inui M, Kobayashi M, Kurusu Y, Yukawa H, Res. Microbiol., 1993, Vol. 144 (3), p181-185).

  A plasmid vector capable of introducing a gene into a coryneform bacterium is not particularly limited as long as it contains at least a gene that controls the replication and proliferation function in the coryneform bacterium. Specific examples thereof include, for example, plasmid pTZ4 described in JP-A-2006-320208; plasmid pC3.14 described in International Publication WO2005-021770; plasmid pCRY30 described in JP-A-3-210184; Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX described in Kaihei 2-72876 and US Pat. No. 5,185,262; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; PAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; pAJ655, pAJ611, and pA described in JP-A-58-192900 1844; described in JP-57-134500 JP pCG1; may be mentioned pCG4 and pCG11 like described in JP 57-183799 JP; pCG2 described in JP 58-35197 JP. Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX are preferably used.

  In the above plasmid vector, the promoter for expressing the alsS gene may be any promoter that functions in coryneform bacteria, and may be the promoter of the alsS gene itself to be used. The expression level of the alsS gene can also be adjusted by appropriately selecting a promoter. Specific examples of promoters that function in coryneform bacteria include TZ4 promoter, fba promoter, tac promoter, trc promoter, and the like.

In addition, in imparting 2,3-butanediol-producing ability, a microorganism modified so as to impart or enhance acetolactate decarboxylase activity may be used.
Here, “acetolactate decarboxylase (hereinafter also referred to as ALDC) activity” refers to an activity (EC: 4.1.1.5) that catalyzes a reaction for producing acetoin from acetolactate. “ALDC activity is enhanced” means that ALDC activity is higher than that of an unmodified strain. The ALDC activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. It was confirmed that ALDC activity was imparted or enhanced by a known method such as the method of Loken et al. (Loke JP, Stormer FC, Eur. J. Biochem., 1970, Vol. 14 (1), p133-137). This can be confirmed by measuring the activity.

A strain modified so as to confer or enhance ALDC activity can be produced in the same manner as the method for conferring or enhancing ALS activity described above.
The alsD gene used for imparting or enhancing ALDC activity is not particularly limited as long as it encodes a protein having ALDC activity, and examples thereof include a gene derived from Bacillus subtilis.
Furthermore, bacteria other than Bacillus subtilis, or alsD genes derived from other microorganisms and animals and plants can also be used. For alsD genes derived from microorganisms or animals and plants, a gene whose base sequence has already been determined or a gene encoding a protein having ALDC activity based on homology, etc., is isolated from the chromosomes of microorganisms, animals and plants, etc. The determined one can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In enhancing ALDC activity, multiple types of alsD genes derived from different microorganisms or animals and plants may be used.

  When ALS activity and ALDC activity are imparted or enhanced, the introduced alsS gene and alsD gene may be present on the same gene locus, or may be present on different gene loci. As an example in which two genes exist on the same locus, for example, an operon formed by linking each gene can be mentioned.

Furthermore, in imparting 2,3-butanediol-producing ability, a microorganism modified so as to impart or enhance 2,3-butanediol dehydrogenase activity may be used.
Here, “2,3-butanediol dehydrogenase (hereinafter also referred to as BDH) activity” means an activity that catalyzes a reaction of reducing acetoin to produce 2,3-butanediol (EC: 1.1.1). .-, 1.1.1.4, 1.1.1.76). “Enhanced BDH activity” means that the BDH activity is higher than that of the unmodified strain. The BDH activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. BDH activity was imparted or enhanced by a known method such as the method of Ui et al. (Ui S, Masuda H, Muraki H, J Ferment. Technol., 1983, Vol. 61 (5), p467-471). This can be confirmed by measuring the BDH activity.

Strains modified to impart or enhance BDH activity can be produced in the same manner as the above-described method for imparting or enhancing ALS activity.
The bdhA gene used for imparting or enhancing BDH activity is not particularly limited as long as it encodes a protein having BDH activity, and examples thereof include a gene derived from Bacillus subtilis.
Furthermore, bacteria other than Bacillus subtilis, or bdhA genes derived from other microorganisms and animals and plants can also be used. A bdhA gene derived from a microorganism or animal or plant is isolated from a chromosome of a microorganism, animal or plant, etc. by isolating a gene whose base sequence has already been determined or a gene encoding a protein having BDH activity based on homology, etc. The determined one can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In enhancing the BDH activity, a plurality of types of bdhA genes derived from different microorganisms or animals and plants may be used.

Furthermore, in imparting 2,3-butanediol-producing ability, it may be a microorganism modified so as to impart or enhance diacetylreductase activity.
Here, “diacetyl reductase activity (hereinafter also referred to as DR)” is an activity that catalyzes a reaction of reducing diacetyl to produce acetoin (EC: 1.1.1.103, 1.1.1.104). ). “DR activity is enhanced” means that DR activity is higher than that of an unmodified strain. The DR activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared with the unmodified strain. DR activity was imparted or enhanced by a known method such as the method of Ui et al. (Ui S, Masuda H, Muraki H, J Ferment. Technol., 1983, Vol. 61 (5), p467-471). This can be confirmed by measuring DR activity.

  Acetolactate produced from pyruvic acid is decarboxylated spontaneously in the presence of oxygen, and diacetyl is produced without requiring enzyme activity. Diacetyl thus produced is reduced by diacetyl reductase and converted to acetoin. This acetoin is further converted into 2,3-butanediol, which can contribute to an improvement in the productivity of 2,3-butanediol.

A strain modified so as to confer or enhance DR activity can be produced in the same manner as the method for conferring or enhancing ALS activity described above.
The bdhA gene used for imparting or enhancing DR activity is not particularly limited as long as it encodes a protein having DR activity, and examples thereof include a gene derived from Bacillus subtilis.
Furthermore, bacteria other than Bacillus subtilis, or bdhA genes derived from other microorganisms and animals and plants can also be used. A bdhA gene derived from a microorganism or animal or plant is isolated from a chromosome of a microorganism, animal or plant, etc. from a gene whose base sequence has already been determined or a gene encoding a protein having DR activity based on homology, etc. The determined one can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In enhancing the DR activity, a plurality of types of bdhA genes derived from different microorganisms or animals and plants may be used.

  In addition, like the enzyme encoded by this bdhA gene, an enzyme having BDH activity often has DR activity. When such a gene is used, BDH activity and DR activity can be simultaneously imparted or enhanced.

  In order to improve the productivity of 2,3-butanediol, the enzyme activity that catalyzes the rate-limiting reaction in the metabolic pathway for biosynthesis of 2,3-butanediol from pyruvate may be imparted or enhanced, Achieved by performing modification that imparts or enhances at least one enzyme activity selected from the group consisting of acetolactate synthase activity, acetolactate decarboxylase activity, 2,3-butanediol dehydrogenase activity, and diacetylreductase activity it can. In order to further improve the productivity of 2,3-butanediol, preferably, two or more types, more preferably three or more types, and particularly preferably modification that enhances all four types of activities. Is desirable.

The microorganism used in the present invention may be a microorganism that inherently has an ability to assimilate xylose or may be one that has been imparted with an ability to assimilate xylose by breeding.
Specific examples of modifications that impart xylose utilization by breeding include modifications that enhance xylose isomerase activity and xylulokinase activity.

  Here, “xylose isomerase (hereinafter also referred to as XylA) activity” refers to an activity (EC: 5.3.1.5) that catalyzes a reaction of isomerizing xylose to produce xylulose. “The XylA activity is enhanced” means that the XylA activity is higher than that of the unmodified strain. The XylA activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. XylA activity was imparted or enhanced by known methods such as the method of Gao et al. (Gao Q, Zhang M, McMillan JD, Kompala DS, Appl. Biochem. Biotechnol., 2002, Vol. 98 (100), p341. -55) can be confirmed by measuring XylA activity.

A strain modified so that XylA activity is imparted or enhanced can be produced in the same manner as described above for imparting or enhancing ALS activity.
The xylA gene used for imparting or enhancing XylA activity is not particularly limited as long as it encodes a protein having XylA activity, and examples thereof include a gene derived from Escherichia coli (Escherichia coli).
Furthermore, bacteria other than E. coli, or xylA genes derived from other microorganisms and animals and plants can also be used. For a xylA gene derived from a microorganism or animal or plant, a gene whose base sequence has already been determined or a gene encoding a protein having XylA activity based on homology or the like is isolated from a chromosome of a microorganism, animal or plant, etc. The determined one can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In enhancing XylA activity, a plurality of types of xylA genes derived from different microorganisms or animals and plants may be used.

In addition, in imparting xylose utilization ability, a microorganism modified so as to impart or enhance xylulokinase activity may be used.
Here, “xylulokinase (hereinafter also referred to as XylB) activity” refers to an activity (EC: 2.7.1.17) that catalyzes a reaction of phosphorylating xylulose to produce xylulose 5-phosphate. . “XylB activity is enhanced” means that XylB activity is higher than that of the unmodified strain. The XylB activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. XylB activity was imparted or enhanced by known methods such as the method of Eliasson et al. (Eliasson A, Boles E, Johansson B, Osternberg M, Thevelin JM, Spencer-Martin I, Juhnh HH). Microbiol.Biotechnol., 2000, Vol.53, p376-82) can be confirmed by measuring XylB activity.

Strains modified so that XylB activity is imparted or enhanced can be produced in the same manner as described above for imparting or enhancing ALS activity.
The xylB gene used for imparting or enhancing XylB activity is not particularly limited as long as it encodes a protein having XylB activity, and examples thereof include genes derived from Escherichia coli and Corynebacterium glutamicum. be able to.
Furthermore, bacteria other than the above, or xylB genes derived from other microorganisms and animals and plants can also be used. For the xylB gene derived from microorganisms or animals and plants, a gene whose base sequence has already been determined or a gene encoding a protein having XylB activity based on homology, etc., is isolated from the chromosomes of microorganisms, animals and plants, etc. The determined one can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. For enhancing XylB activity, a plurality of types of xylB genes derived from different microorganisms or animals and plants may be used.

  When XylA activity and XylB activity are imparted or enhanced, the introduced xylA gene and xylB gene may be present on the same locus, or may be present on different loci. As an example in which two genes exist on the same locus, for example, an operon formed by linking each gene can be mentioned.

  The microorganism of the present invention is further modified as described above to further reduce by-products (such as PoxB activity and PC activity) and to impart or enhance 2,3-butanediol-producing ability ( (ALS activity, ALDC activity, BDH activity, enhancement of DR activity, etc.) Microorganism obtained by combining two or more types of alterations for imparting xylose utilization (XylA activity, enhancement of XylB activity, etc.) It may be. When making a plurality of modifications, the order does not matter.

<Method for producing 2,3-butanediol>
The production method of the present invention includes a step of producing 2,3-butanediol by causing the microorganism of the present invention or a processed product thereof to act on an organic raw material in an aqueous medium (hereinafter referred to as “fermentation step”). .

In the production method of the present invention, a processed product of the microorganism of the present invention can also be used. Examples of the processed microorganisms include, for example, immobilized microorganisms in which microorganisms are immobilized with acrylamide, carrageenan, etc., disrupted microorganisms, centrifuged supernatants thereof, or supernatants thereof by ammonium sulfate treatment, etc. Examples include partially purified fractions.
Moreover, it is preferable that the manufacturing method of this invention has the process (henceforth a "recovery process") which collects the produced 2, 3- butanediol after the said fermentation process.

When the microorganism of the present invention is used in the production method of the present invention, a slant cultured medium such as an agar medium may be directly used. However, prior to the fermentation step, the microorganism may be preliminarily used in a liquid medium. What was cultured may be used. That is, the fermentation process can be performed after the microorganisms of the present invention are proliferated in advance by performing seed culture and main culture described later.
In addition, the seed culture and main culture which will be described later and the fermentation process which will be described later can be performed simultaneously without distinction. In addition, 2,3-butanediol can be produced by reacting with an organic raw material while growing a microorganism cultured in seed culture or main culture in a reaction solution.

(Seed culture)
The seed culture is performed in order to prepare the cells of the microorganism of the present invention to be subjected to the main culture. As a medium used for seed culture, a normal medium used for culturing microorganisms can be used, but a medium containing a nitrogen source, an inorganic salt, or the like is preferable. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and grown by the microorganism of the present invention. Specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein degradation product, peptone And various organic and inorganic nitrogen compounds such as yeast extract, meat extract and corn steep liquor. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary.

  In seed culture, a carbon source may be added to the medium as necessary. The carbon source used for seed culture is not particularly limited as long as the microorganism can be assimilated and proliferated. Usually, carbohydrates such as galactose, lactose, glucose, fructose, xylose, arabinose, sucrose, starch, and cellulose; Fermentable carbohydrates such as polyalcohols such as glycerol, mannitol, inositol and ribitol are used, among which glucose, fructose, sucrose, xylose or arabinose are preferable, and glucose, sucrose or xylose is particularly preferable. These carbon sources may be added alone or in combination.

  The seed culture is preferably performed at a general optimum growth temperature. The general optimum growth temperature means a temperature at which the growth rate is fastest under the conditions used for production of 2,3-butanediol. The specific culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. In the case of coryneform bacteria, the temperature is usually 25 ° C to 35 ° C, more preferably 28 ° C to 33 ° C, and particularly preferably about 30 ° C.

  The seed culture is preferably performed at a general optimum pH for growth. The general optimum growth pH refers to a pH having the fastest growth rate under the conditions used for the production of organic compounds. The specific culture pH is usually pH 4 to 10, preferably pH 6 to 8. In the case of coryneform bacteria, the pH is usually 6-9, with pH 6.5-8.5 being preferred.

  The culture time for seed culture is not particularly limited as long as a certain amount of cells can be obtained, but is usually 6 hours or more and 96 hours or less. In the seed culture, oxygen is preferably supplied by aeration or stirring.

  The bacterial cells after the seed culture can be used for the main culture described later. However, the seed culture may be omitted, and the cells cultured on the slope with a solid medium such as an agar medium may be directly used for the main culture. Moreover, you may repeat seed culture several times as needed.

(Main culture)
The main culture is performed in order to prepare the microbial cells of the microorganism of the present invention to be used in the 2,3-butanediol production reaction described later, and the main purpose is to increase the amount of the microbial cells. When performing the seed culture described above, the main culture is performed using the cells obtained by seed culture.

  The medium used for the main culture can be a normal medium used for culturing microorganisms, but is preferably a medium containing a nitrogen source, an inorganic salt, or the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and propagated by the present microorganism. Specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein degradation product, peptone, yeast Various organic and inorganic nitrogen compounds such as extract, meat extract and corn steep liquor can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. In order to suppress foaming during culture, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.

  In the main culture, it is preferable to add a carbon source to the medium. The carbon source used in the main culture is not particularly limited as long as the microorganism can be assimilated and proliferated. Usually, carbohydrates such as galactose, lactose, glucose, fructose, xylose, arabinose, sucrose, starch, and cellulose; Fermentable carbohydrates such as polyalcohols such as glycerol, mannitol, inositol and ribitol are used, among which glucose, fructose, sucrose, xylose or arabinose are preferable, and glucose, sucrose or xylose is particularly preferable. These carbon sources may be added alone or in combination.

Moreover, the starch saccharified liquid containing the said fermentable saccharide | sugar, molasses etc. are also used, and what is the saccharide liquid which the said fermentable saccharide extracted from plants, such as sugarcane, sugar beet, and a sugar maple, is preferable.
These carbon sources may be added alone or in combination.

  The concentration of the carbon source used is not particularly limited, but it is advantageous to add it within a range that does not inhibit the growth of microorganisms, and is usually 0.1 to 10% (W / V), preferably 0, relative to the culture solution. 0.5 to 5% (W / V) can be used. Further, a carbon source may be additionally added in accordance with the decrease of the carbon source accompanying the growth.

  The main culture is preferably performed at a general optimum growth temperature. The specific culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. In the case of coryneform bacteria, the temperature is usually 25 ° C to 35 ° C, more preferably 28 ° C to 33 ° C, and particularly preferably about 30 ° C.

  Further, the main culture is preferably performed at a general optimum growth pH. The specific culture pH is usually pH 4 to 10, preferably pH 6 to 8. In the case of coryneform bacteria, the pH is usually 6-9, with pH 6.5-8.5 being preferred.

  The culture time for the main culture is not particularly limited as long as a certain amount of cells can be obtained, but is usually 6 hours or more and 96 hours or less. In the main culture, it is preferable to supply oxygen by aeration or stirring.

  In addition, in the main culture, as a method for preparing cells more suitable for the production of 2,3-butanediol, the carbon source depletion and sufficiency described in JP-A-2008-259451 are alternately repeated in a short time. A method of culturing can also be used.

  The cells after the main culture can be used for 2,3-butanediol production reaction, which will be described later, but the culture solution may be used directly or after the cells are collected by centrifugation, membrane separation, etc. Also good.

(Fermentation process)
In the fermentation process, 2,3-butanediol is produced by allowing the above-mentioned microorganism having 2,3-butanediol-producing ability or a processed product thereof to act on an organic raw material in an aqueous medium. The reaction that occurs in this fermentation process is hereinafter referred to as “2,3-butanediol production reaction”.

  Here, the aqueous medium is an aqueous solution that performs a 2,3-butanediol production reaction in the fermentation process, and is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like as described later. In the aqueous medium, 2,3-butanediol production reaction can be performed by reacting the microorganism of the present invention or a processed product thereof with an organic raw material. In this specification, the aqueous medium means all liquids contained in the reaction vessel.

  The aqueous medium may be, for example, a medium for culturing microorganisms or a buffer solution such as a phosphate buffer, but the reaction solution is an aqueous solution containing a nitrogen source, an inorganic salt, or the like. It is preferable. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by the microorganism of the present invention to produce 2,3-butanediol, and specifically, ammonium salt, nitrate, urea, soybean hydrolysate. Various organic and inorganic nitrogen compounds such as degradation products, casein degradation products, peptone, yeast extract, meat extract, corn steep liquor and the like can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. In order to suppress foaming during the reaction, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the reaction solution.

The organic raw material used in the present invention is not particularly limited as long as the microorganism of the present invention can be assimilated to produce 2,3-butanediol, and so-called general carbohydrates can be used.
Specifically, monosaccharides having 3 carbon atoms (triose) such as glyceraldehyde; monosaccharides having 4 carbon atoms (tetrose) such as erythrose, threose, erythrulose; ribose, lyxose, xylose, arabinose, deoxyribose, xylulose 5 carbon monosaccharides (pentose) such as ribulose; 6 carbon atoms such as allose, talose, growth, glucose, altrose, mannose, galactose, idose, fucose, fucose, rhamnose, psicose, fructose, sorbose, tagatose Monosaccharide (hexose); monosaccharides having 7 carbon atoms such as cedoheptulose (heptose); disaccharides such as sucrose, lactose, maltose, trehanose, tulanose, cellobiose; trisaccharides such as raffinose, melezitose, maltotriose; Rakutoorigo sugars, galactooligosaccharides, oligosaccharides such Man'naorigo sugar; starch, dextrin, cellulose, hemicellulose, glucans, polysaccharides pentosan like; glycerol, mannitol, inositol, polyalcohols such as ribitol, and the like.

Among the sugars described above, a sugar containing a monosaccharide having 3 to 7 carbon atoms as a constituent component is preferable, and among these, at least selected from the group consisting of hexose, pentose, and a disaccharide containing these as constituent components. One type is more preferable.
As hexose, glucose, fructose, mannose and galactose are preferable, and glucose is more preferable. As pentose, xylose and arabinose are preferable, and xylose is more preferable. As the disaccharide containing hexose and pentose as components, sucrose is preferable. This is because glucose, xylose, and sucrose are the main constituents of nature and plants, and therefore it is easy to obtain raw materials.
Note that the organic raw material used in the present invention may contain one kind of sugar alone or two or more kinds of sugars.

  The organic raw material used in the present invention is not particularly limited as long as it contains the saccharide. For example, one or more saccharides dissolved in water to form an aqueous solution constitute one or more saccharides. A plant body containing a component or a part thereof decomposed to a carbohydrate, a plant body containing one or more types of the above-mentioned carbohydrates as a constituent component, or a substance obtained by extracting a carbohydrate from the part can be used. Specifically, a lignocellulose decomposition raw material, a sucrose-containing raw material, a starch decomposition raw material, and the like, which will be described later, may be mentioned.

The organic raw material used in the present invention may be used by diluting with water or the like to lower the saccharide concentration as necessary, or may be concentrated to increase the saccharide concentration.
The concentration of the saccharide contained in the organic raw material in the present invention is not particularly limited because it varies greatly depending on the origin of the organic raw material, the type of the saccharide contained, etc., but productivity of the fermentation production process and the chemical conversion process Is usually 0.1% by mass or more, preferably 2% by mass or more, and usually 80% by mass or less, preferably 70% by mass or less. However, when two or more kinds of carbohydrates are included, the total concentration is shown.

A preferable organic raw material is a lignocellulose decomposition raw material.
Lignocellulose is an organic substance composed of structural polysaccharide cellulose, hemicellulose, and polymer lignin of an aromatic compound. Lignocellulose is usually not edible and is usually discarded or incinerated, and is preferable in that it can be supplied stably and resources can be used effectively.
Lignocellulose decomposition materials include herbaceous biomass such as bagasse, corn stover, wheat straw, rice straw, switchgrass, napiergrass, Eliansus, Sasa and Susuki, and woody biomass such as waste wood, sawdust, bark and waste paper Etc. can be used suitably. Of these, bagasse, corn stover, and straw are preferable.

  The method for obtaining the organic raw material from the above lignocellulose-decomposing raw material is not particularly limited. For example, after pretreatment of lignocellulose as necessary, it is performed with an enzyme, an acid, subcritical water, supercritical water, or the like. Examples of the method include hydrolysis or thermal decomposition.

Moreover, a sucrose containing raw material is mentioned as a preferable organic raw material.
Further, sucrose is included in plants that can accumulate sucrose in cells. Hereinafter, such plants are referred to as “plants containing sucrose”. Examples of plants containing sucrose include those used as raw materials for sugar such as sugar cane, sugar beet, sugar maple, sugar beet, and sorghum, among which sugar cane and sugar beet are preferred.

  Although the method of obtaining an organic raw material from the plant containing sucrose is not specifically limited, For example, the method of squeezing or leaching after pulverizing the said plant etc. are mentioned. In the present invention, plant juice containing sucrose thus obtained (for example, cane juice in the case of sugarcane), crude sugar, molasses and the like can also be used as organic raw materials.

Moreover, a starch decomposition raw material is mentioned as a preferable organic raw material.
In addition, starch is included in plants that can accumulate starch in cells, and such plants are hereinafter referred to as “plants containing starch”. Examples of the plant containing starch include cassava, corn, potato, wheat, sweet potato, sago palm, rice, scrap, peanut, mung bean, bracken, green lily, etc. Among them, cassava, corn, potato and wheat are preferable.

  Although the method of obtaining an organic raw material from the plant containing starch is not specifically limited, For example, the method etc. which hydrolyze the starch extracted from the said plant are mentioned.

  The concentration of the organic raw material used is not particularly limited, but it is advantageous and preferable in terms of productivity if it is as high as possible within a range not inhibiting the production of 2,3-butanediol. The concentration of the organic raw material contained in the aqueous medium is usually 5% (W / V) or more, preferably 10% (W / V) or more, based on the concentration of the saccharide contained therein, with respect to the aqueous medium. On the other hand, it is usually 30% (W / V) or less, preferably 20% (W / V) or less. Moreover, you may add by adding an organic raw material according to the reduction | decrease of the said organic raw material accompanying progress of the production reaction of 2, 3- butanediol.

  The pH of the aqueous medium during the 2,3-butanediol production reaction is preferably adjusted to a range where the activity is most effectively exhibited according to the type of the microorganism of the present invention to be used. Specifically, when coryneform bacteria are used, the pH of the reaction solution is usually 5.0 or higher, preferably 5.5 or higher, more preferably 6.0 or higher, and still more preferably 6.5 or higher. On the other hand, it is usually 10 or less, preferably 9.5 or less, more preferably 9.0 or less.

  When the by-product produced during the 2,3-butanediol production reaction is an acidic substance, the pH of the aqueous medium is sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, ammonium carbonate, It can be adjusted by adding ammonium bicarbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia (ammonium hydroxide), or a mixture thereof. If the by-product produced during the 2,3-butanediol production reaction is a basic substance, add an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, an organic acid such as acetic acid, or a mixture thereof. Or by supplying carbon dioxide gas.

  The amount of microbial cells used in the 2,3-butanediol production reaction is not particularly limited, but is usually 1 g / L or more, preferably 10 g / L or more, more preferably 20 g / L or more as the weight of wet cells. On the other hand, it is usually 700 g / L or less, preferably 500 g / L or less, more preferably 400 g / L or less.

  The time for the 2,3-butanediol production reaction is not particularly limited, but is usually 1 hour or longer, preferably 3 hours or longer, and is usually 168 hours or shorter, preferably 72 hours or shorter.

  The temperature of the 2,3-butanediol production reaction may be performed at the same temperature as the optimum growth temperature of the microorganism to be used, but it is advantageous to carry out the reaction at a temperature higher than the optimum growth temperature. It is preferable to carry out at a temperature as high as 7 ° C, preferably 7 ° C to 15 ° C. Specifically, in the case of coryneform bacteria, it is usually 30 ° C. or higher, preferably 35 ° C. or higher, more preferably 39 ° C. or higher, and usually 45 ° C. or lower, preferably 43 ° C. or lower, more preferably 41 It is below ℃. During the organic compound production reaction, it is not always necessary to set the temperature within the range of 30 ° C. to 45 ° C. However, it is desirable to set the temperature within the above temperature range for 50% or more, preferably 80% or more of the total reaction time.

  The 2,3-butanediol production reaction may be carried out in an anaerobic atmosphere in which oxygen is not supplied but is preferably aerated and stirred. Specifically, the oxygen transfer rate is usually 0 mmol / L / h or more, preferably 10 mmol / L / h or more, more preferably 20 mmol / L / h or more, while usually 200 mmol / L / h or less, preferably Is 150 mmol / L / h or less, more preferably 100 mmol / L / h or less.

  The method for producing 2,3-butanediol of the present invention is not particularly limited, but can be applied to any of batch reaction, semi-batch reaction, and continuous reaction.

(Recovery process)
In the present invention, 2,3-butanediol is produced by the above-mentioned 2,3-butanediol production reaction and can be accumulated in the reaction solution. The accumulated 2,3-butanediol may further include a step of recovering from the aqueous medium according to a conventional method. Specifically, for example, solid substances such as bacterial cells are removed by centrifugation, filtration, and the like, and high purity 2,3-butanediol can be recovered by distillation after the nanofiltration membrane treatment.
The obtained 2,3-butanediol can be used as a raw material for pharmaceutical and cosmetic intermediate materials, printing inks, fragrances, plasticizers, softeners, insecticides and the like.

<Method for producing 1,3-butadiene>
In the present invention, after producing 2,3-butanediol by the method described above, 1,3-butadiene can be produced according to a conventional method using the obtained 2,3-butanediol as a raw material. Specifically, for example, a method of detaching acetic acid after acetylation can be mentioned.
1,3-Butadiene is a starting material capable of synthesizing many kinds of compounds such as hexamethylenediamine, adipic acid, 1,4-butanediol, etc. Since there is a possibility that an existing synthetic resin can be replaced with biomass, its production technology has attracted attention.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[Production Example 1]
About Brevibacterium flavum MJ233 strain, lactate dehydrogenase gene (ldh gene), phosphotransacetylase gene (pta gene), acetate kinase gene (ack gene), and CoA transferase gene (ctf gene) In addition, the acetolactate synthase gene (alsS gene), the acetolactate decarboxylase gene (alsD), and the 2,3-butanediol dehydrogenase gene (bdhA gene) are introduced, and the Brevibacterium flavum MJ233 / BdhA / AlsSD is introduced. The strain / ΔCTF / ΔPTA-ACK / ΔLDH was prepared.

<Preparation of PTA-ACK disruption strain>
Brevibacterium flavum MJ233 / ΔLDH strain (LDH disruption strain: WO2005-021770) was used as a test strain for preparing the PTA-ACK disruption strain. Plasmid DNA used for transformation of the strain was prepared from Escherichia coli JM110 strain transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159) using pTACK1 (Japanese Patent Laid-Open No. 2008-0667629).
Transformation of Brevibacterium flavum MJ233 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was LBG agar medium containing 50 μg / mL kanamycin. [Tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water].
Since the strain grown on this medium is a plasmid in which pTACK1 cannot be replicated in the Brevibacterium flavum MJ233 strain, the same gene on the genome of the pta-ack gene of the plasmid and the Brevibacterium flavum MJ233 strain As a result of homologous recombination with the kanamycin, the kanamycin resistance gene and SacB gene derived from the plasmid should be inserted on the same genome.

Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 50 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, several tens of strains that were considered to be sucrose-insensitive due to the loss of the SacB gene by the second homologous recombination were obtained.
Among the strains thus obtained, those in which the pta-ack gene has been replaced with a mutant derived from pTACK1 and those in which the wild type has been restored are included. Confirmation of whether the pta-ack gene is a mutant type or a wild type can be easily performed by subjecting the cells obtained by culturing in the LBG medium directly to the PCR reaction and detecting the pta-ack gene. I can confirm. When the pta-ack gene is analyzed using primers (SEQ ID NOs: 1 and 2) for PCR amplification, a DNA fragment of 1,645 bp is generated in the wild type and 1,086 bp in the mutant type having the deletion region. As a result of analyzing the sucrose-insensitive strain by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔPTA-ACK / ΔLDH.

<Preparation of CTF disruption strain>
As the test strain for preparing the CTF disruption strain, the Brevibacterium flavum MJ233 / ΔPTA-ACK / ΔLDH strain prepared above was used. Plasmid DNA used for transformation of the strain was prepared from E. coli strain JM110 transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159) using pACH22 (JP 2008-0667629).
Transformation of Brevibacterium flavum MJ233 / ΔPTA-ACK / ΔLDH strain and selection of twice homologous recombinants were carried out in the same manner as the preparation of the PTA-ACK-disrupted strain, and several tens of sucrose insensitive A colony was obtained. Of these, the determination of whether the ctf gene is a mutant type or a wild type can be carried out by analyzing the ctf gene using primers (SEQ ID NOs: 3 and 4) for PCR amplification. In this case, a DNA fragment of 1,228 bp is generated, and a mutant having a deletion region of 1,003 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔCTF / ΔPTA-ACK / ΔLDH.

<Preparation of ALS, ALDC, BDH enhanced strain>
(A) Construction of expression vector using Fba promoter Plasmid vectors capable of protein expression in coryneform bacteria using the Fba promoter derived from Brevibacterium flavum MJ233 strain are pTZ4 vector (Japanese Patent Application Laid-Open No. 2006-320208) and pC3. .14 vector (WO2005-021770) was prepared by inserting a DNA fragment obtained by amplifying the region of the promoter sequence of fructose 1,6-bisphosphate aldolase. The Fba promoter sequence was synthesized by a gene synthesis service (Genscript) based on a sequence upstream of the gene (GenBank Database Accession No. AP005276) of Corynebacterium glutamicum ATCC13032 strain for which the entire genome sequence was reported (SEQ ID NO: 5).
Using this as a template, the Fba promoter region was amplified by PCR using the synthesized primers (SEQ ID NOs: 6 and 7). Composition of reaction solution: 5 μL of template DNA, 0.3 μL of PrimeStar DNA polymerase (Takara Bio Inc.), 10 μL of 5-fold concentration attached buffer, 0.3 μL of primer for each 100 μM, and 4 μL of attached dNTPs were mixed to a total volume of 50 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 54 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 5 times, followed by 94 ° C. for 15 seconds. The cycle of 60 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 25 times. Furthermore, the heat retention at 94 ° C. was added for 3 minutes before the cycle, and the heat retention at 72 ° C. for 3 minutes was added after the final cycle.
The obtained DNA fragment of the Fba promoter region was purified using QIAquick PCR Purification Kit (Qiagen) and then cleaved with restriction enzymes KpnI and PstI or restriction enzymes KpnI and ApaI. The resulting DNA fragments of about 0.2 kb were separated by 0.9% agarose gel electrophoresis and then visualized by ethidium bromide staining, and then gelled using a MonoFas DNA purification kit (GL Science). Recovered from.

  These DNA fragments were mixed with DNA prepared by cutting the coryneform bacterial expression vector pC3.14 with restriction enzymes KpnI and PstI, and the coryneform bacterial expression vector pTZ4 with KpnI and ApaI, and DNA Ligation Kit Mighty Mix (Takara). Biotechnology). Escherichia coli HTS08 strain (Takara Bio Inc.) was transformed with each of the obtained plasmid DNAs, and the former was smeared on an LB agar medium containing 25 μg / mL spectinomycin and the latter was 50 μg / mL kanamycin. The obtained clone was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (the former containing 25 μg / mL spectinomycin, the latter containing 50 μg / mL kanamycin), and then QIAprep Spin Miniprep Kit (Qiagen). Was used to purify each plasmid DNA. Each of the obtained plasmid DNAs was confirmed to have an Fba promoter sequence introduced using primers (SEQ ID NOs: 8 and 9, the latter were SEQ ID NOs: 9 and 10), and these were named pC3.14F and pTZ4F, respectively. .

(B) Construction of plasmid for enhancing AlsSD The alsS gene and alsD gene derived from Bacillus subtilis (Bacilli subtilis ATCC6051 strain) constitute an operon (hereinafter also referred to as alsSD), and both genes were cloned in this structure. It was.
Bacillus subtilis (strain Bacillus subtilis ATCC6051) is cultured in LB medium [Yeast Extract 5 g / L, Bacto trypton 10 g / L, sodium chloride 10 g / L] until the late logarithmic growth phase, Invitrogen ChargeSwitch gDNAterMiB Genomic DNA was prepared according to the protocol. Genomic DNA was diluted 1000 times with sterile water and used as a PCR template.
Obtaining an alsSD gene fragment derived from Bacillus subtilis (Bacilli subtilis ATCC6051) strain using the DNA prepared above as a template, genome sequence information (GenBank Database Accession No. AL009126.3) for which the entire nucleotide sequence has already been reported, and PCR was performed using primers (SEQ ID NOs: 11 and 12) designed based on the sequence information of the gene (GenBank Database Accession No. L04470.1). Composition of reaction solution: 5 μL of template DNA, 0.3 μL of PrimeStar DNA polymerase (Takara Bio Inc.), 10 μL of 5-fold concentration attached buffer, 0.3 μL of primer for each 100 μM, and 4 μL of attached dNTPs were mixed to a total volume of 50 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 54 ° C. for 30 seconds and 72 ° C. for 2 minutes was repeated 5 times, followed by 94 ° C. for 15 seconds, 60 The cycle of 30 ° C. for 30 seconds and 72 ° C. for 2 minutes was repeated 25 times. Furthermore, the heat retention at 94 ° C. was added for 3 minutes before the cycle, and the heat retention at 72 ° C. for 3 minutes was added after the final cycle.
The obtained DNA fragment of the alsSD gene sequence was purified using QIAquick PCR Purification Kit (Qiagen) and then cleaved with restriction enzymes BamHI and ApaI. The resulting DNA fragment of about 2.5 kb was separated by 0.9% agarose gel electrophoresis and then visualized by ethidium bromide staining and recovered from the gel using the MonoFas DNA purification kit (GL Science). did.

  This DNA fragment was mixed with DNA prepared by cleaving pC3.14F constructed in (A) above with restriction enzymes BamHI and ApaI, and ligated using DNA Ligation Kit Mighty Mix (Takara Bio Inc.). Escherichia coli HTS08 strain (Takara Bio Inc.) was transformed with the obtained plasmid DNA, and smeared on an LB agar medium containing 25 μg / mL spectinomycin. The obtained clone was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL spectinomycin), and then the plasmid DNA was purified using QIAprep Spin Miniprep Kit (Qiagen). did. The obtained plasmid DNA was cleaved with restriction enzymes BamHI and ApaI to confirm that an inserted fragment of about 2.5 kb was confirmed, and this was named pC3.14F-AlsSD. (The construction procedure is shown in FIG. 2.)

(C) Construction of plasmid for enhancing BdhA The bdhA gene fragment derived from Bacillus subtilis (Bacillus subtilis ATCC6051) strain was obtained using the Bacillus subtilis genomic DNA prepared above as a template, and the genome sequence information for which the entire nucleotide sequence has already been reported (GenBank Database Accession No. AL009126.3) and PCR using primers (SEQ ID NOs: 13 and 14) designed based on the sequence information of the gene (GenBank Database Accession No. CAB12443.1). Composition of reaction solution: 5 μL of template DNA, 0.3 μL of PrimeStar DNA polymerase (Takara Bio Inc.), 10 μL of 5-fold concentration attached buffer, 0.3 μL of primer for each 100 μM, and 4 μL of attached dNTPs were mixed to a total volume of 50 μL. Reaction temperature condition: DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 54 ° C. for 30 seconds and 72 ° C. for 1 minute was repeated 5 times, followed by 94 ° C. for 15 seconds, 60 The cycle of 30 ° C. for 30 seconds and 72 ° C. for 1 minute was repeated 25 times. Furthermore, the heat retention at 94 ° C. was added for 3 minutes before the cycle, and the heat retention at 72 ° C. for 3 minutes was added after the final cycle.
The obtained DNA fragment of the bdhA gene sequence was purified using QIAquick PCR Purification Kit (Qiagen) and then cleaved with restriction enzymes BamHI and ApaI. The resulting 1.1 kb DNA fragment was separated by 0.9% agarose gel electrophoresis and then visualized by ethidium bromide staining and recovered from the gel using the MonoFas DNA purification kit (GL Science). did.

  This DNA fragment was mixed with DNA prepared by cleaving pTZ4F constructed in (A) above with restriction enzymes BamHI and ApaI, and ligated using DNA Ligation Kit Mighty Mix (Takara Bio Inc.). Escherichia coli HTS08 strain (Takara Bio Inc.) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kanamycin. The obtained clone was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (including 50 μg / mL kanamycin), and then the plasmid DNA was purified using QIAprep Spin Miniprep Kit (Qiagen). The obtained plasmid DNA was cleaved with restriction enzymes BamHI and ApaI to confirm that an inserted fragment of about 1.1 kb was confirmed, and this was named pTZ4F-BdhA. (The construction procedure is shown in FIG. 3)

(D) Production of AlsSD and BdhA-enhanced strains The test strains for producing AlsSD and BdhA-enhanced strains were the Brevibacterium flavum MJ233 / ΔCTF / ΔPTA-ACK / ΔLDH strain produced above. In order to use this strain for transformation, the calcium chloride method (Journal of Molecular Biology, 1970, 53) using pC3.14F-AlsSD constructed in (B) and pTZ4F-BdhA constructed in (C), respectively. , 159) was used to transform E. coli JM110 strain to prepare demethylated plasmid DNA.
Brevibacterium flavum MJ233 / ΔCTF / ΔPTA-ACK / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the resulting transformant was 50 μg / mL. LBG agar medium containing kanamycin and 10 μg / mL streptomycin [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L distilled water] was smeared. In the strain grown on this medium, both pC3.14F-AlsSD and pTZ4F-BdhA are maintained in the Brevibacterium flavum MJ233 strain, and this strain is stored in Brevibacterium flavum MJ233 / BdhA / AlsSD / It was named ΔCTF / ΔPTA-ACK / ΔLDH (hereinafter also referred to as MBD04).

[Production Example 2]
For Brevibacterium flavum MJ233 strain, lactate dehydrogenase gene (ldh gene), phosphotransacetylase gene (pta gene), acetate kinase gene (ack gene), CoA transferase gene (ctf gene), and Disrupting the pyruvate oxidase gene (poxB gene) and introducing the acetolactate synthase gene (alsS gene), the acetolactate decarboxylase gene (alsD), and the 2,3-butanediol dehydrogenase gene (bdhA gene) A bacterial flavum MJ233 / BdhA / AlsSD / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH strain was prepared.

<Preparation of PoxB disruption strain>
The Brevibacterium flavum MJ233 / ΔCTF / ΔPTA-ACK / ΔLDH strain prepared in Preparation Example 1 was used as a test strain for preparation of the PoxB-disrupted strain. Plasmid DNA used for transformation of the strain was prepared from E. coli strain JM110 transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159) using pOXB11 (Japanese Patent Laid-Open No. 2008-0667629).
Transformation of Brevibacterium flavum MJ233 / ΔCTF / ΔPTA-ACK / ΔLDH strain and selection of twice homologous recombinants were carried out in the same manner as the preparation of the PTA-ACK disrupted strain, and several tens of sucrose Insensitive colonies were obtained. Of these, determination of whether the poxB gene is a mutant type or a wild type can be performed by analyzing the poxB gene using primers (SEQ ID NOs: 15 and 16) for PCR amplification. In this case, a DNA fragment of 1,518 bp is generated, and in a mutant having a deletion region, a DNA fragment of 981 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH. .

<Preparation of ALS, ALDC, BDH enhanced strain>
As the test strains for producing AlsSD and BdhA-enhanced strains, the Brevibacterium flavum MJ233 / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH strain prepared above was used. Transformation of the strain was carried out in the same manner as in the preparation of AlsSD and BdhA-enhanced strains in Preparation Example 1, and the prepared strain was selected from Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH (hereinafter referred to as “BrevA”) , Also called MBD05).

[Production Example 3]
For the Brevibacterium flavum MJ233 strain, lactate dehydrogenase gene (ldh gene), phosphotransacetylase gene (pta gene), acetate kinase gene (ack gene), CoA transferase gene (ctf gene), pill Disruption of the beet oxidase gene (poxB gene) and the pyruvate carboxylase gene (pc gene), as well as the acetolactate synthase gene (alsS gene), the acetolactate decarboxylase gene (alsD), and the 2,3-butanediol dehydrogenase gene ( bdhA gene), and Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPC / ΔPoxB / ΔCTF / ΔPTA-A To produce a K / ΔLDH strain.

<Production of PC disruption strain>
(E) Construction of a plasmid for PC disruption PC coding region consisting of about 0.4 kb by cleaving plasmid pMJPC17.2 (WO2012-128231) for replacing pc gene promoter on the genome with restriction enzymes BamHI and BglII The amino terminal sequence of was excised. The remaining DNA fragment of about 5.9 kb was separated by 0.9% agarose gel electrophoresis, and then recovered using a QIAquick Gel Extraction Kit (Qiagen). The ends of the recovered DNA fragments were labeled with DNA Ligation Kit ver. 2 (Takara Bio) was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin. The obtained clone was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL kanamycin), and then a plasmid DNA was prepared using QIAprep Spin Miniprep Kit (Qiagen). As a result of cleaving the thus obtained plasmid DNA with the restriction enzyme KpnI, a fragment showing a deletion of about 2.0 kb on the amino terminal side was confirmed, and this was named pKB-PC1.

(F) Production of PC-enhanced strain As a test strain for producing a PC-disrupted strain, the Brevibacterium flavum MJ233 / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH strain prepared in Preparation Example 1 was used. The plasmid DNA used for transformation of the strain was prepared from E. coli JM110 strain transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159) using pKB-PC1 constructed in (E) above. .
Transformation of Brevibacterium flavum MJ233 / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH strain and selection of twice homologous recombinants were carried out in the same manner as in the preparation of the above-mentioned PTA-ACK disrupted strain. Sucrose-insensitive colonies were obtained. Of these, the determination of whether the pc gene is a mutant type or a wild type can be made by analyzing the pc gene using primers (SEQ ID NOs: 17 and 18) for PCR amplification. In this case, a DNA fragment of 1,297 bp is generated, and a mutant having a deletion region of 1,203 bp is generated. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was selected as Brevibacterium flavum MJ233 / ΔPC / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH. Named.

<Preparation of ALS, ALDC, BDH enhanced strain>
As the test strains for producing AlsSD and BdhA-enhanced strains, the Brevibacterium flavum MJ233 / ΔPC / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH strain prepared above was used. Transformation of the strain was carried out in the same manner as the preparation of the AlsSD and BdhA-enhanced strain of Preparation Example 1, and the prepared strain was used as Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPC / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH. (Hereinafter also referred to as MBD06).

[Production Example 4]
For the Brevibacterium flavum MJ233 strain, the acetolactate synthase gene (alsS gene), the acetolactate decarboxylase gene (alsD), and the 2,3-butanediol dehydrogenase gene (bdhA gene) are introduced as follows. Brevibacterium flavum MJ233 / BdhA / AlsSD strain was prepared.

<Preparation of ALS, ALDC, BDH enhanced strain>
Test strains for preparing AlsSD and BdhA-enhanced strains were prepared by conventional methods (Wolf Hetal, J. Bacteriol., 1983, 156 (3), p1165-70, Kurusu Yetal., Agric Biol Chem., 1990, 54 (2), Brevibacterium flavum MJ233 strain from which the endogenous plasmid was removed (curing) according to p443-7) was used. Transformation of the strain was performed in the same manner as the preparation of the AlsSD and BdhA-enhanced strain of Preparation Example 1, and the prepared strain was named Brevibacterium flavum MJ233 / BdhA / AlsSD (hereinafter also referred to as MBD01).

[Production Example 5]
For the Brevibacterium flavum MJ233 strain, disruption of the lactate dehydrogenase gene (ldh gene), the acetolactate synthase gene (alsS gene), the acetolactate decarboxylase gene (alsD), and 2,3- A butanediol dehydrogenase gene (bdhA gene) was introduced to prepare a Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔLDH strain.

<Preparation of ALS, ALDC, BDH enhanced strain>
Brevibacterium flavum MJ233 / ΔLDH strain (LDH disruption strain: WO2005-021770) was used as a test strain for preparing AlsSD and BdhA-enhanced strains. Transformation of the strain was performed in the same manner as the preparation of the AlsSD and BdhA-enhanced strain in Preparation Example 1, and the prepared strain was named Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔLDH (hereinafter also referred to as MBD02). .

[Production Example 6]
For the Brevibacterium flavum MJ233 strain, disruption of the lactate dehydrogenase gene (ldh gene), the phosphotransacetylase gene (pta gene), and the acetate kinase gene (ack gene), and the acetolactate synthase gene (AlsS gene), acetolactate decarboxylase gene (alsD), and 2,3-butanediol dehydrogenase gene (bdhA gene) were introduced, and Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPTA-ACK / ΔLDH strain was obtained. Produced.

<Preparation of ALS, ALDC, BDH enhanced strain>
The Brevibacterium flavum MJ233 / ΔPTA-ACK / ΔLDH strain prepared in Preparation Example 1 was used as a test strain for preparing the AlsSD and BdhA-enhanced strain. Transformation of the strain was carried out in the same manner as the production of the AlsSD and BdhA-enhanced strain of Example 1, and the produced strain was designated as Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPTA-ACK / ΔLDH (hereinafter also referred to as MBD03). ).

[Example 1]
<Proliferation evaluation of MBD04 strain>
In order to evaluate the growth of the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔCTF / ΔPTA-ACK / ΔLDH (MBD04) strain prepared in Preparation Example 1, a bacterial cell was prepared by performing seed culture as follows. Then, flask culture was implemented as main culture.

(A) Seed culture In order to perform seed culture, CGXII medium (ammonium sulfate 20 g / L, urea 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, MOPS 42 g / L, magnesium sulfate. Heptahydrate 0.25 g / L, calcium chloride 10 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate pentahydrate 10 mg / L, zinc sulfate heptahydrate 1 mg / L , Copper sulfate 0.2 mg / L, nickel chloride hexahydrate 0.02 mg / L, biotin 0.2 mg / L, glucose 40 g / L, protocatechuic acid 30 mg / L, pH 7.0) 2 mL test Add 2 μL each of sterile filtered 50 mg / ml kanamycin aqueous solution and 25 mg / ml streptomycin aqueous solution to the MBD04 strain. Whole day and night and, with shaking cultured at 32 ℃.

(B) Main culture In order to carry out the main culture, 50 ml of CGXII medium (containing kanamycin and streptomycin as described above) was placed in a 200 ml normal flask, and the seed culture obtained so that the absorbance at OD ₆₆₀ was 0.1. The solution was added and cultured with shaking at 30 ° C. After 24 hours from the start of culture, 2.5 ml of an 80% (w / v) glucose solution was added. Further, sampling was performed at the stage of 48 hours and 69 hours after the start of culture, and the absorbance of OD ₆₆₀ was measured.
As a result, the value of OD ₆₆₀ of 48 hours after the start of culture 13.1, the value of OD ₆₆₀ after culture after 69 hours was 14.7.

[Example 2]
<Proliferation evaluation of MBD05 strain>
The same procedure as in Example 1 was carried out except that the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH (MBD05) strain prepared in Preparation Example 2 was used. As a result, the value of OD ₆₆₀ of 48 hours after the start of culture 13.9, the value of OD ₆₆₀ after culture after 69 hours was 14.2.

[Example 3]
<Evaluation of growth of MBD06 strain>
The same procedure as in Example 1 was carried out except that the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPC / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH (MBD06) strain prepared in Preparation Example 3 was used. As a result, the value of OD ₆₆₀ of 48 hours after the start of culture 14.7, the value of OD ₆₆₀ after culture after 69 hours was 16.4.

[Comparative Example 1]
<Proliferation evaluation of MBD01 strain>
The same procedure as in Example 1 was conducted except that the Brevibacterium flavum MJ233 / BdhA / AlsSD (MBD01) strain prepared in Preparation Example 4 was used. As a result, the value of OD ₆₆₀ of 48 hours after the start of culture 17.1, the value of OD ₆₆₀ after culture after 69 hours was 16.1.

[Comparative Example 2]
<Proliferation evaluation of MBD02 strain>
The same procedure as in Example 1 was conducted except that the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔLDH (MBD02) strain prepared in Preparation Example 5 was used. As a result, the value of OD ₆₆₀ of 48 hours after the start of culture 9.34, the value of OD ₆₆₀ after culture after 69 hours was 9.50.

[Comparative Example 3]
<Proliferation evaluation of MBD03 strain>
The same procedure as in Example 1 was carried out except that the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPTA-ACK / ΔLDH (MBD03) strain prepared in Preparation Example 6 was used. As a result, the value of OD ₆₆₀ after 48 hours from the start of culture was 10.6, and the value of OD ₆₆₀ after 69 hours from the start of culture was 11.0.

As a result of Examples 1 to 3 and Comparative Examples 1 to 3, OD ₆₆₀ values 48 hours and 69 hours after the start of culture are summarized in Table 1.

From Table 1, the growth rate of the MBD02 strain in which the ldh gene was disrupted compared to the MBD01 strain, which is a 2,3-butanediol-producing strain, was greatly reduced as compared with the MBD01 strain, and compared with the reached OD ₆₆₀ after 69 hours of culture. It turned out to be about 59%. Further, MBD03 strain that destroyed more pta-ack gene against MBD02 strain, although slightly growth rate than MBD02 strain is restored, remained improved by about 16% compared with reach OD ₆₆₀ after incubation for 69 hours It was.
On the other hand, MBD04 strain that destroyed more ctf gene against MBD03 strain, growth rate significantly recovered compared to MBD02 strain, was improved by about 55% compared with reach OD ₆₆₀ after incubation for 69 hours. The MBD05 strain in which the poxB gene was further disrupted relative to the MBD04 strain had a growth rate equivalent to that of the MBD04 strain. The MBD06 strain in which the pc gene was further disrupted compared to the MBD05 strain recovered the growth rate more than the MBD04 strain, and improved by about 73% compared to the MBD02 strain compared with the reached OD ₆₆₀ after 69 hours of culture. Recovered to the same level.
As described above, in the 2,3-butanediol-producing strain, when the ldh gene is disrupted, the growth is deteriorated, but by destroying the pta-ack gene and the ctf gene, the growth rate is significantly recovered. It was revealed that the strain recovered to the same level as the strain of. These genetic modifications are important not only for reducing by-products but also for maintaining the growth ability of 2,3-butanediol-producing strains.

[Example 4]
<Production evaluation of MBD05 strain for 2,3-butanediol>
In order to evaluate 2,3-butanediol production of Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH (MBD05) strain prepared in Preparation Example 2, seed culture was performed as follows. Then, the cells were prepared, and then 2,3-butanediol production reaction was carried out simultaneously with the main culture.

(G) Seed culture In order to perform seed culture, CGXII medium (ammonium sulfate 20 g / L, urea 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, MOPS 42 g / L, magnesium sulfate. Heptahydrate 0.25 g / L, calcium chloride 10 mg / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate pentahydrate 10 mg / L, zinc sulfate heptahydrate 1 mg / L , Copper sulfate 0.2 mg / L, nickel chloride hexahydrate 0.02 mg / L, biotin 0.2 mg / L, glucose 40 g / L, protocatechuic acid 30 mg / L, pH 7.0) 2 mL test 2 μL each of sterile filtered 50 mg / ml kanamycin aqueous solution and 25 mg / ml streptomycin aqueous solution were added to the MBD05 strain. Whole day and night and, with shaking cultured at 32 ℃.

(H) 2,3-butanediol production reaction In order to carry out the 2,3-butanediol production reaction simultaneously with the main culture, 50 ml of CGXII medium (containing kanamycin and streptomycin as described above) was placed in a 200 ml normal flask, and OD _The seed culture solution obtained in (G) above was added so that the absorbance at ₆₆₀ was 0.2, and the mixture was cultured with shaking at 30 ° C. 2.5 ml of an 80% (w / v) glucose solution was added 24 hours after the start of the culture and 42 hours later. In addition, sampling was performed at the stage of 40 hours and 64 hours after the start of culture, and the culture supernatant was collected after centrifugation, diluted 10-fold with distilled water, and analyzed by liquid chromatography and gas chromatography.
Each analysis condition is as follows. Lactic acid, acetic acid and succinic acid are quantified by liquid chromatography analysis, and acetoin, (Meso) -2,3-butanediol, (R, R) or (S, S) -2,3-butanediol are gas chromatographed. Quantified by analysis.

(Liquid chromatography analysis conditions)
Pump: Hitachi High-Technologies Corporation L-2130
Column oven: Hitachi High-Technologies L-2350
UV detector: Hitachi High-Technologies Corporation L-2400
RI detector: L-2490 manufactured by Hitachi High-Technologies Corporation
Column: ULTRON PS-80H 8.0ID × 300mmL manufactured by Shinwa Kako Co., Ltd.
Temperature: 60 ° C
Eluent: 0.11% by mass perchloric acid solution 1.0 mL / min Detection method: UV (210 nm), RI
Injection volume: 10 μL
(Gas chromatography analysis conditions)
Device: GC17A manufactured by Shimadzu Corporation
Column: DB-17 (0.25 mmφ × 30 m film thickness 0.25 μm)
carrier: He 1.0 mL / min
Split ratio: 1/50
Inlet temperature: 260 ° C
Detector temperature: 280 ° C
Column temperature: 50 ° C. → 280 ° C. 10 ° C./min
Detector: FID
Injection volume: 1 μL

  As a result, 40 hours after the start of the reaction, lactic acid was 0.41 g / L, acetic acid was 1.21 g / L, succinic acid was 4.67 g / L, acetoin was 2.10 g / L, (Meso) -2,3- Butanediol is 1.69 g / L, (R, R) or (S, S) -2,3-butanediol is 9.08 g / L, lactic acid 64 hours after the start of the reaction is 0.75 g / L, and acetic acid is 1.27 g / L, succinic acid 1.55 g / L, acetoin 3.87 g / L, (Meso) -2,3-butanediol 3.33 g / L, (R, R) or (S, S ) -2,3-butanediol was 12.4 g / L.

[Example 5]
<Evaluation of 2,3-butanediol production by MBD06 strain>
It was carried out in the same manner as in Example 4 except that the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔPC / ΔPoxB / ΔCTF / ΔPTA-ACK / ΔLDH (MBD06) strain prepared in Preparation Example 3 was used. As a result, 40 hours after the start of the reaction, lactic acid was 0.22 g / L, acetic acid was 1.16 g / L, succinic acid was 4.29 g / L, acetoin was 1.11 g / L, (Meso) -2,3- 1.49 g / L for butanediol, 11.0 g / L for (R, R) or (S, S) -2,3-butanediol, 0.58 g / L for lactic acid 64 hours after the start of the reaction, 1.24 g / L, succinic acid 1.24 g / L, acetoin 2.82 g / L, (Meso) -2,3-butanediol 2.79 g / L, (R, R) or (S, S ) -2,3-butanediol was 13.4 g / L.

[Comparative Example 4]
<Production evaluation of MBD01 strain for 2,3-butanediol>
The same procedure as in Example 4 was performed except that the Brevibacterium flavum MJ233 / BdhA / AlsSD (MBD01) strain prepared in Preparation Example 4 was used. As a result, lactic acid 40 hours after the start of the reaction was 11.4 g / L, acetic acid was 1.02 g / L, succinic acid was 1.32 g / L, acetoin was 2.82 g / L, (Meso) -2,3- Butanediol is 1.02 g / L, (R, R) or (S, S) -2,3-butanediol is 9.87 g / L, lactic acid 64 hours after the start of the reaction is 11.6 g / L, and acetic acid is 1.03 g / L, succinic acid 0.04 g / L, acetoin 4.16 g / L, (Meso) -2,3-butanediol 1.11 g / L, (R, R) or (S, S ) -2,3-butanediol was 10.2 g / L.

[Comparative Example 5]
<Production evaluation of MBD02 strain for 2,3-butanediol>
The same procedure as in Example 4 was performed except that the Brevibacterium flavum MJ233 / BdhA / AlsSD / ΔLDH (MBD02) strain prepared in Preparation Example 5 was used. As a result, 40 hours after the start of the reaction, lactic acid was 0.12 g / L, acetic acid was 5.18 g / L, succinic acid was 1.90 g / L, acetoin was 0.60 g / L, (Meso) -2,3- Butanediol is 0.53 g / L, (R, R) or (S, S) -2,3-butanediol is 4.32 g / L, lactic acid after 64 hours of reaction is 0.36 g / L, and acetic acid is 5.22 g / L, succinic acid 0.15 g / L, acetoin 1.15 g / L, (Meso) -2,3-butanediol 0.95 g / L, (R, R) or (S, S ) -2,3-butanediol was 4.86 g / L.

  As a result of Examples 4 and 5 and Comparative Examples 4 and 5, lactic acid, acetic acid, succinic acid, acetoin, (Meso) -2,3-butanediol, 40 hours and 64 hours after the start of culture, (R, R ) Or (S, S) -2,3-butanediol and 2,3-butanediol total values are summarized in Table 2.

From Table 2, MBD02 strain in which the ldh gene is disrupted with respect to MBD01 strain, which is a 2,3-butanediol-producing strain, significantly reduces the by-product of lactic acid compared to MBD01 strain, but 2,3-butanediol It was found that the productivity of ((Meso), (R, R), (S, S)) was also reduced, and the accumulated concentration after 64 hours of reaction was about 51%. This is considered to be due to the fact that the ldh gene was disrupted, resulting in a decrease in the growth rate and a lack of bacterial mass. In addition, the MBD02 strain increased the amount of acetic acid and succinic acid by-products compared to the MBD01 strain.
On the other hand, the MBD05 strain in which the pta-ack gene, the ctf gene, and the poxB gene were further disrupted compared to the MBD02 strain had a significantly reduced by-product of acetic acid compared to the MBD02 strain, and compared with the accumulated concentration after 64 hours of reaction As a result, a reduction of about 76% was achieved. In addition, the productivity of 2,3-butanediol (total of (Meso), (R, R), (S, S)) is greatly improved, and compared to the MBD01 strain compared with the accumulated concentration after 64 hours of reaction. About 39%. However, the by-product of succinic acid increased more than MBD02 strain.
In the MBD06 strain in which the pc gene was further disrupted compared to the MBD05 strain, succinic acid by-product was reduced as compared with the MBD05 strain, and a reduction of about 20% was achieved compared with the accumulated concentration after 64 hours of reaction. In addition, the productivity of 2,3-butanediol (total of (Meso), (R, R), (S, S)) is further improved, and compared to the MBD01 strain compared with the accumulated concentration after 64 hours of reaction. About 43%.
As described above, in the 2,3-butanediol-producing strain, by-production of lactic acid, acetic acid, and succinic acid can be reduced by destroying ldh gene, pta-ack gene, ctf gene, poxB gene, and pc gene. It was shown that the productivity of 2,3-butanediol can be improved.

ADVANTAGE OF THE INVENTION According to this invention, the production | generation of the 2, 3- butanediol can be provided by reducing the production | generation of a by-product without accompanying the remarkable fall of the growth rate of microorganisms with progress of fermentation.
By using such a microorganism of the present invention, 2,3-butanediol can be produced efficiently and safely. Therefore, the present invention has high industrial applicability.

Claims (10)

A microorganism having an ability to produce 2,3-butanediol, and modified so that the following activities a), b), and c) are reduced as compared with an unmodified strain.
a) Lactate dehydrogenase activity b) Acetate kinase activity and / or phosphotransacetylase activity c) CoA transferase activity   The microorganism according to claim 1, which has been modified so that pyruvate oxidase activity is reduced compared to an unmodified strain.   The microorganism according to claim 1 or 2, which has been modified so that pyruvate carboxylase activity is reduced as compared to an unmodified strain.   Further, at least one enzyme activity selected from the group consisting of acetolactate synthase activity, acetolactate decarboxylase activity, 2,3-butanediol dehydrogenase activity, and diacetylreductase activity is enhanced as compared with an unmodified strain. The microorganism according to any one of claims 1 to 3, which has been modified.   Furthermore, the microorganism as described in any one of Claims 1-4 modified | changed so that xylose isomerase activity might be increased compared with an unmodified strain.   Furthermore, the microorganism as described in any one of Claims 1-5 modified | changed so that xylulokinase activity might be increased compared with an unmodified strain.   The microorganism is a coryneform bacterium, Escherichia coli, Bacillus genus bacteria, Anaerobiospirillum genus bacteria, Actinobacillus genus bacteria, Mannheimia genus bacteria, Basfia moth The microorganism according to any one of claims 1 to 6, which is at least one selected from the group consisting of (Zymomonas) bacteria, Zymobacter bacteria, filamentous fungi, and yeasts.   A method for producing 2,3-butanediol, comprising causing the microorganism according to any one of claims 1 to 7 or a processed product thereof to act on an organic raw material in an aqueous medium.   The method for producing 2,3-butanediol according to claim 8, wherein the organic raw material contains one or more selected from the group consisting of glucose, sucrose, and xylose.   A step of producing 2,3-butanediol by the method according to claim 8 or 9, and a step of converting into 1,3-butadiene using the 2,3-butanediol obtained in the step as a raw material. A method for producing 1,3-butadiene.

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