2,3-butanediol-producing transformant and method for producing 2,3-butanediol using said transformant

Abstract

The purpose of the present invention is to provide a transformant having excellent 2,3-butanediol production capability. Provided is a transformant of a hydrogen-oxidizing bacterium that grows in the co-presence of oxygen, the transformant being used for the production of 2,3-butanediol. In the transformant, (a) a nucleic acid encoding an enzyme for synthesizing acetolactate from pyruvic acid, (b) a nucleic acid encoding an enzyme for converting acetolactate into acetoin, (c) a nucleic acid encoding an enzyme for converting acetoin into 2,3-butanediol, and (d) a regulatory sequence operably linked to the nucleic acids (a) to (c) are introduced, wherein the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c).

Description

2,3-Butanediol-producing transformant and method for producing 2,3-butanediol using said transformant

[0001] The present invention relates to a 2,3-butanediol-producing transformant, a method for producing 2,3-butanediol using the transformant, a vector for producing the transformant, and a transformation method using the vector.

[0002] 2,3-butanediol is a commodity chemical used in the production of various chemical products. For example, 2,3-butanediol is used in a variety of fields as a raw material for 1,3-butadiene and methyl ethyl ketone, as a solvent, chain extender, dispersant, and as a raw material for printing inks, moisturizing agents in cosmetics, fragrances, plasticizers, pharmaceutical intermediates, liquid crystals, insecticides, fabric softeners, antifreeze, and food products. 2,3-butanediol used industrially is mainly produced by organic chemical methods using crude oil as a raw material.

[0003] From the perspective of the global environment, bioprocesses using microbial fermentation have attracted attention as a way to shift from crude oil to biomass as a raw material. Many microorganisms are known to naturally produce 2,3-butanediol from carbohydrates through fermentation, but they have drawbacks such as the need for biomass pretreatment, the possibility of being pathogenic, and low 2,3-butanediol productivity.

[0004] Meanwhile, bioprocesses using chemoautotrophic microorganisms capable of producing substances using CO2 directly as a carbon source have also attracted attention due to their potential for fixing large amounts of carbon. For example, a method is known in which useful substances are synthesized from CO2 by genetically modifying hydrogen-oxidizing bacteria with high CO2 fixation capacity. Non-Patent Document 1 describes the production of 2,3-butanediol by expressing the alsS gene, alsD gene, and adh gene, which are involved in 2,3-butanediol synthesis, in an arabinose-inducible system.

[0005] RR Bommareddy et al., A Sustainable Chemicals Manufacturing Paradigm Using CO2 and Renewable H2, iScience, 2020 June 26, Vol 23, 101218

[0006] The genetically modified strain produced in Non-Patent Document 1 does not produce a sufficient amount of 2,3-butanediol. Furthermore, in Non-Patent Document 1, the acoXABC gene cluster, which corresponds to the enzyme group involved in acetoin decomposition and which is inherent to hydrogen-oxidizing bacteria, is deleted in order to increase the amount of 2,3-butanediol produced. The genetic modification requires two steps: a step of recombining the 2,3-butanediol synthesis genes and a step of deleting the acoXABC gene cluster.

[0007] Therefore, there is a need for the development of a bioprocess using a genetic recombinant that can be produced more easily and more efficiently to produce 2,3-butanediol.

[0008] An object of the present invention is to provide a microorganism capable of producing 2,3-butanediol in high yield.

[0009] The present invention relates to a transformant of a hydrogen-oxidizing bacterium that grows in the presence of oxygen, which is used for producing 2,3-butanediol, and into which the following have been introduced: (a) a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate; (b) a nucleic acid encoding an enzyme that converts acetolactate to acetoin; (c) a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol; and (d) a regulatory sequence operably linked to the nucleic acids (a) to (c), wherein the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c).

[0010] The hydrogen-oxidizing bacteria are preferably hydrogen-oxidizing bacteria capable of synthesizing pyruvic acid using carbonate ions and / or CO2 as a carbon source.

[0011] It is preferable that the hydrogen-oxidizing bacteria be at least one species selected from the group consisting of hydrogen-oxidizing bacteria belonging to the genus Cupriavidus, hydrogen-oxidizing bacteria belonging to the genus Hydrogenophilus, hydrogen-oxidizing bacteria belonging to the genus Hydrogenobacter, and hydrogen-oxidizing bacteria belonging to the genus Hydrogenovibrio.

[0012] The hydrogen-oxidizing bacteria are preferably hydrogen-oxidizing bacteria belonging to the genus Cupriavidus or hydrogen-oxidizing bacteria belonging to the genus Hydrogenophilus.

[0013] It is preferable that the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c), and that the nucleic acid (a) is located upstream of the nucleic acid (c).

[0014] Preferably, the regulatory sequence comprises an inducible promoter.

[0015] It is preferable that by introducing the regulatory sequence and the nucleic acids (a) to (c), at least a partial region of the nucleic acid encoding the enzyme group that synthesizes hydroxyalkanoic acid in the genomic DNA is removed.

[0016] The nucleic acid (a) is preferably a nucleic acid derived from a microorganism belonging to the genus Bacillus or Acinetobacter.

[0017] The nucleic acid (b) is preferably a nucleic acid derived from a microorganism belonging to the genus Bacillus or Enterobacter.

[0018] The nucleic acid (c) is preferably a nucleic acid derived from a microorganism belonging to the genus Clostridium or Acinetobacter.

[0019] It is preferable that the nucleic acid (b) comprises a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or 14, or a nucleic acid having at least 80% identity to the base sequence shown in SEQ ID NO: 1 or 14 and encoding a protein having catalytic activity for producing acetoin from acetolactate.

[0020] In addition, the nucleic acid (b) may be a nucleic acid that has at least 90% identity with the base sequence shown in SEQ ID NO: 1 or 14, or has a base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence of SEQ ID NO: 1 or 14, and encodes a protein that has catalytic activity to produce acetoin from acetolactate.

[0021] It is preferable that the nucleic acid (a) comprises a nucleic acid comprising the base sequence shown in SEQ ID NO: 2 or 15, or a nucleic acid which has at least 80% identity to the base sequence shown in SEQ ID NO: 2 or 15 and encodes a protein having catalytic activity for producing acetolactate from pyruvate.

[0022] The nucleic acid (a) may be a nucleic acid that has at least 90% identity with the base sequence shown in SEQ ID NO: 2 or 15, or has a base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence of SEQ ID NO: 2 or 15, and encodes a protein that has catalytic activity to produce acetolactate from pyruvate.

[0023] It is preferable that the nucleic acid (c) comprises a nucleic acid comprising the base sequence shown in SEQ ID NO: 3 or 16, or a nucleic acid which has at least 80% identity with the base sequence shown in SEQ ID NO: 3 or 16 and encodes a protein having catalytic activity for producing 2,3-butanediol from acetoin.

[0024] In addition, the nucleic acid (c) may be a nucleic acid that has at least 90% identity with the base sequence shown in SEQ ID NO: 3 or 16, or has a base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence of SEQ ID NO: 3 or 16, and encodes a protein that has catalytic activity to produce 2,3-butanediol from acetoin.

[0025] The hydrogen-oxidizing bacteria is preferably Cupriavidus necator.

[0026] Transformants that retain the endogenous acoXABC gene cluster are preferred.

[0027] Preferably, the hydrogen-oxidizing bacterium is a transformant in which the acoXABC gene cluster is at least partially deleted or inactivated.

[0028] The hydrogen-oxidizing bacterium is preferably a transformant in which the acoXABC gene cluster has been deleted.

[0029] The present invention relates to a vector comprising a nucleic acid sequence including a gene capable of expressing an enzyme that synthesizes acetolactate from pyruvate, a gene capable of expressing an enzyme that converts acetolactate to acetoin, and a gene capable of expressing an enzyme that converts acetoin to 2,3-butanediol, wherein the nucleic acid sequence is operably linked to a regulatory sequence for expression of the three enzymes in a hydrogen-oxidizing bacterium, and the gene capable of expressing the enzyme that converts acetolactate to acetoin in the nucleic acid sequence is located upstream of the gene capable of expressing the enzyme that synthesizes acetolactate from pyruvate and the gene capable of expressing the enzyme that converts acetoin to 2,3-butanediol.

[0030] The present invention relates to a transformation method comprising transforming a hydrogen-oxidizing bacterium with the vector according to the present invention.

[0031] The present invention relates to a method for producing 2,3-butanediol, which comprises a step of culturing the transformant according to the present invention.

[0032] The culture is preferably carried out by introducing CO 2 , hydrogen, and oxygen into a medium containing the transformant.

[0033] The transformant of the present invention is a transformant into which exogenous (a) nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, (b) nucleic acid encoding an enzyme that converts acetolactate to acetoin, and (c) nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol have been introduced, wherein the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c). By using such a transformant, 2,3-butanediol can be produced much more efficiently using CO2 directly as a carbon source than by transformants of the prior art.

[0034] Fig. 1 is a diagram schematically showing the structure of a transformant prepared in an example. Fig. 2 is a diagram schematically showing the structure of a transformant prepared in a comparative example. Fig. 3 is a diagram schematically showing the structure of a transformant prepared in an example. Fig. 4 is a diagram schematically showing the structure of a transformant prepared in an example. Fig. 5 is a diagram schematically showing the structure of a transformant prepared in an example.

[0035] The transformant of the present invention is a transformant of a hydrogen-oxidizing bacterium into which (a) a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, (b) a nucleic acid encoding an enzyme that converts acetolactate to acetoin, and (c) a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol have been introduced, and the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c), and is a transformant used for producing 2,3-butanediol.

[0036] The metabolic pathway involved in the biosynthesis of 2,3-butanediol in 2,3-butanediol-producing microorganisms is known to involve three steps, starting with pyruvate obtained from sugar metabolism. In the first step, the thiamine-dependent enzyme α-acetolactate synthase catalyzes the condensation of two molecules of pyruvate to produce α-acetolactate and release CO2. Next, α-acetolactate is converted to acetoin by a decarboxylation reaction catalyzed by α-acetolactate decarboxylase. Finally, acetoin is reduced to 2,3-butanediol by acetoin dehydrogenase using NADH as a cofactor.

[0037] In the present invention, the hydrogen-oxidizing bacteria used as a host are not particularly limited as long as they can grow under autotrophic conditions in the presence of oxygen. Hydrogen-oxidizing bacteria generally have high growth potential and significantly higher energy efficiency in inorganic carbon fixation, making them preferred hosts for producing chemical products from carbon dioxide as a raw material. Any bacterium that synthesizes pyruvic acid as a metabolic intermediate in sugar metabolism using carbonate ions and / or CO2 as a carbon source can be suitably used as the hydrogen-oxidizing bacteria of the present invention. Preferably, the hydrogen-oxidizing bacteria are bacteria capable of producing hydroxyalkanoic acids such as polyhydroxybutyrate (PHB) from pyruvic acid. The PHB synthesis pathway in such hydrogen-oxidizing bacteria is known and involves three major enzymatic steps: conversion of acetyl-CoA to acetoacetyl-CoA by β-ketothiolase (acetyl-CoA C-acetyltransferase, phaA), reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by NADPH-dependent acetoacetyl-CoA reductase (PhaB), and synthesis of PHB from 3-hydroxybutyryl-CoA by PHA synthase (PhaC).

[0038] Such hydrogen-oxidizing bacteria include those belonging to the genus Cupriavidus, those belonging to the genus Hydrogenophilus, those belonging to the genus Hydrogenobacter, and microorganisms belonging to the genus Hydrogenovibrio. Preferably, the hydrogen-oxidizing bacteria of the present invention are those belonging to the genus Cupriavidus or those belonging to the genus Hydrogenophilus. For example, Cupriavidus necator is a preferred microorganism belonging to the genus Cupriavidus because of its adaptability to diverse environments.

[0039] In the present invention, hydrogen-oxidizing bacteria can be modified (e.g., genetically engineered) by any method known in the art so as to contain and / or express at least a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, a nucleic acid encoding an enzyme that converts acetolactate to acetoin, and a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol. A transformant of the modified hydrogen-oxidizing bacteria is a bacterium that is capable of producing 2,3-butanediol even when cultured using carbon dioxide and / or carbonate ions as the sole carbon source. Note that "bacteria capable of producing 2,3-butanediol" refers to a bacterium that, when cultured in a medium, has the ability to secrete 2,3-butanediol into the medium and produce 2,3-butanediol to an extent that it can be recovered from the medium.

[0040] The transformant of the present invention comprises at least three nucleic acids, namely, a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, a nucleic acid encoding an enzyme that converts acetolactate to acetoin, and a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol, as well as regulatory sequences operably linked to these nucleic acids. In the present invention, the regulatory sequence is operably linked upstream of the at least three nucleic acids and may include a transcription promoter. "Operably linked" refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. "Operably linked" means that the linked DNA sequences are usually contiguous, and if necessary, the linking of one or more protein-coding regions is contiguous and in reading frame.

[0041] Preferably, the transcription promoter is an inducible promoter. The at least three nucleic acids of the present invention can be integrated into the genomic DNA of a host organism in a state where they can be expressed under the control of an inducible promoter in the host organism.

[0042] In one embodiment, the nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, the nucleic acid encoding an enzyme that converts acetolactate to acetoin, and the nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol are operably linked to a regulatory sequence including at least an arabinose-inducible promoter as an inducible promoter and a regulatory gene araC encoding the arabinose-responsive regulatory protein AraC. The arabinose-inducible promoter may be, for example, the araBAD promoter.

[0043] In the present invention, for example, a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate is an acetolactate synthase gene. From the viewpoint of 2,3-butanediol productivity, the alsS gene is preferred. Examples include the alsS gene or its orthologue derived from a microorganism belonging to the genus Bacillus, or the alsS gene or its orthologue derived from a microorganism belonging to the genus Acinetobacter. More preferably, for example, the alsS gene may be the alsS gene of Bacillus subtilis or its orthologue, or the alsS gene of Acinetobacter venetianus or its orthologue, with the alsS gene of Bacillus subtilis or its orthologue being particularly preferred. For example, the base sequence of the alsS gene of Bacillus subtilis includes a sequence containing the base sequence shown in SEQ ID NO: 2. For example, the base sequence of the alsS gene of Acinetobacter venetianus includes a sequence containing the base sequence shown in SEQ ID NO: 15.

[0044] Examples of nucleic acids encoding enzymes having acetolactate synthase activity in the present invention include nucleic acids of (i) or (ii) below: (i) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 2 or 15. (ii) a nucleic acid having a nucleotide sequence in which one or more nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2 or 15, which nucleic acid has at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identity to the nucleotide sequence shown in SEQ ID NO: 2 or 15 and encodes a protein having catalytic activity for producing acetolactate from pyruvate.

[0045] The nucleic acid encoding an enzyme having acetolactate synthase activity in the present invention may also be, for example, a nucleic acid having a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2 or 15, which has a nucleotide sequence encoding an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence encoded by SEQ ID NO: 2 or 15, and which encodes a protein having catalytic activity for synthesizing acetolactate from pyruvate, or a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2 or 15. Note that "stringent conditions" generally refer to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. Such stringent conditions are well known to those skilled in the art and can be determined by referring to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).

[0046] In the present invention, for example, a nucleic acid encoding an enzyme that converts acetolactate to acetoin is an acetolactate decarboxylase gene. From the viewpoint of 2,3-butanediol productivity, the alsD gene is preferred. Examples include the alsD gene or its orthologue derived from a microorganism belonging to the genus Bacillus, or the alsD gene or its orthologue derived from a microorganism belonging to the genus Enterobacter. More preferably, the alsD gene may be, for example, the alsD gene of Bacillus subtilis or its orthologue, or the alsD gene of Enterobacter kobei or its orthologue. The alsD gene of Bacillus subtilis or its orthologue is particularly preferred. For example, an example of the nucleotide sequence of the alsD gene of Bacillus subtilis is a sequence containing the nucleotide sequence set forth in SEQ ID NO: 1. For example, the nucleotide sequence of the alsD gene of Enterobacter kobei includes a sequence containing the nucleotide sequence shown in SEQ ID NO:14.

[0047] Examples of nucleic acids encoding enzymes having acetolactate decarboxylase activity in the present invention include nucleic acids of (iii) or (iv) below: (iii) A nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 1 or 14. (iv) A nucleic acid having a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 1 or 14, which nucleic acid has at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identity to the nucleotide sequence shown in SEQ ID NO: 1 or 14 and encodes a protein having catalytic activity for converting acetolactate to acetoin.

[0048] The nucleic acid encoding an enzyme having acetolactate decarboxylase activity in the present invention may also be, for example, a nucleic acid having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 1 or 14, which has a base sequence encoding an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence encoded by SEQ ID NO: 1 or 14, and which encodes a protein having catalytic activity to produce acetoin from acetolactate, or a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a base sequence complementary to a nucleic acid having the base sequence shown in SEQ ID NO: 1 or 14.

[0049] In the present invention, for example, a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol is an acetoin dehydrogenase gene. From the viewpoint of 2,3-butanediol productivity, the adh gene is preferred. Examples include an adh gene derived from a microorganism belonging to the genus Clostridium or its orthologue, or an adh gene derived from a microorganism belonging to the genus Acinetobacter or its orthologue. More preferably, for example, the adh gene may be the adh gene of Clostridium beijerinckii or its orthologue, or the adh gene of Acinetobacter lactucae or its orthologue, and the adh gene of Clostridium beijerinckii or its orthologue is particularly preferred. For example, the base sequence of the adh gene of Clostridium beijerinckii includes a sequence containing the base sequence shown in SEQ ID NO: 3. For example, the base sequence of the adh gene of Acinetobacter lactucae includes a sequence containing the base sequence shown in SEQ ID NO: 16.

[0050] Examples of nucleic acids encoding an enzyme having acetoin dehydrogenase activity in the present invention include nucleic acids of (v) or (vi) below: (v) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 3 or 16. (vi) a nucleic acid having a nucleotide sequence in which one or more bases have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 3 or 16, which nucleic acid has at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identity to the nucleotide sequence shown in SEQ ID NO: 3 or 16 and encodes a protein having catalytic activity for converting acetoin to 2,3-butanediol.

[0051] The nucleic acid encoding an enzyme having acetolactate decarboxylase activity in the present invention may also be, for example, a nucleic acid having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 3 or 16, which has a base sequence encoding an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to the amino acid sequence encoded by SEQ ID NO: 3 or 16, and which encodes a protein having catalytic activity for converting acetoin to 2,3-butanediol, or a nucleic acid that hybridizes under stringent conditions to a nucleic acid having a base sequence complementary to the nucleic acid having the base sequence shown in SEQ ID NO: 3 or 16.

[0052] In the present invention, the term "orthologous" gene refers to a related gene that exists in a different organism and encodes a protein with a homologous function.

[0053] In the transformant of the present invention, a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, a nucleic acid encoding an enzyme that converts acetolactate to acetoin, and a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol, as well as regulatory sequences operably linked to these nucleic acids, can be incorporated so as to remove at least a partial region of the gene encoding the enzymes that synthesize hydroxyalkanoic acids in the genomic DNA of the hydrogen-oxidizing bacterium. This removal eliminates or reduces the function of the enzymes required for hydroxyalkanoic acid synthesis. As a result, the synthesis system from pyruvate to, for example, PHB via acetyl-CoA is disrupted or reduced in the hydrogen-oxidizing bacterium, and the enzymes for synthesizing 2,3-butanediol from pyruvate are expressed.

[0054] For example, in hydrogen-oxidizing bacteria of the genus Capriavidus, the phaC1AB1 operon is constructed, encoding PHA synthase (PhaC1), acetyl-CoA C-acetyltransferase (PhaA), and acetoacetyl-CoA reductase (PhaB1), which are key enzymes in the PHB synthesis pathway. Therefore, preferably, a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate, a nucleic acid encoding an enzyme that converts acetolactate to acetoin, and a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol, as well as regulatory sequences operably linked to these nucleic acids, are integrated into the genome of the host so that at least a portion of the phaC1AB1 operon is deleted from the genome. Preferably, the nucleic acid encoding the enzyme that synthesizes acetolactate from pyruvate, the nucleic acid encoding the enzyme that converts acetolactate to acetoin, and the nucleic acid encoding the enzyme that converts acetoin to 2,3-butanediol, as well as the regulatory sequences operably linked to these nucleic acids, are integrated into the genome by homologous recombination, with the region upstream of the start codon of the phaC1 gene and the region downstream of the stop codon of the phaB1 gene serving as homologous recombination sequences. The phaC1AB1 operon is knocked out in the host hydrogen-oxidizing bacterium, and the nucleic acid encoding the enzyme that synthesizes acetolactate from pyruvate for 2,3-butanediol production, the nucleic acid encoding the enzyme that converts acetolactate to acetoin, and the nucleic acid encoding the enzyme that converts acetoin to 2,3-butanediol are knocked in.

[0055] In the transformant of the present invention, nucleic acid (a) encoding an enzyme that synthesizes acetolactate from pyruvate, nucleic acid (b) encoding an enzyme that converts acetolactate to acetoin, and nucleic acid (c) encoding an enzyme that converts acetoin to 2,3-butanediol are introduced so that nucleic acid (b) encoding the enzyme that converts acetolactate to acetoin is located upstream of nucleic acid (a) encoding the enzyme that synthesizes acetolactate from pyruvate and nucleic acid (c) encoding the enzyme that converts acetoin to 2,3-butanediol. Surprisingly, the present inventors have discovered for the first time that, for the genes encoding the enzymes involved in the 2,3-butanediol biosynthetic pathway that are placed downstream of regulatory sequences, the transformant can produce significantly higher amounts of 2,3-butanediol by arranging the genes in an order that differs from the order of the steps in the synthesis pathway.

[0056] In the transformant of the present invention, it is desirable that the nucleic acid (a) encoding the enzyme that synthesizes acetolactate from pyruvate, which is the first step in the 2,3-butanediol biosynthetic pathway, is not located at the position closest to the regulatory sequence among the three nucleic acids.

[0057] When nucleic acid (b) encoding an enzyme that converts acetolactate to acetoin is located upstream of nucleic acid (a) encoding an enzyme that synthesizes acetolactate from pyruvic acid and nucleic acid (c) encoding an enzyme that converts acetoin to 2,3-butanediol, nucleic acid (a) encoding an enzyme that synthesizes acetolactate from pyruvic acid may be upstream of nucleic acid (c) encoding an enzyme that converts acetoin to 2,3-butanediol, or nucleic acid (c) encoding an enzyme that converts acetoin to 2,3-butanediol may be upstream of nucleic acid (a) encoding an enzyme that synthesizes acetolactate from pyruvic acid, but it is particularly desirable that nucleic acid (a) encoding an enzyme that synthesizes acetolactate from pyruvic acid be upstream of nucleic acid (c) encoding an enzyme that converts acetoin to 2,3-butanediol.

[0058] As described in the Examples below, a transformant was designed so that the genes encoding each metabolic enzyme involved in the 2,3-butanediol biosynthetic pathway were located in the following order downstream, starting from a position close to the regulatory sequence: nucleic acid (b) encoding an enzyme that converts acetolactate to acetoin, i.e., the alsD gene; nucleic acid (a) encoding an enzyme that synthesizes acetolactate from pyruvate, i.e., the alsS gene; and nucleic acid (c) encoding an enzyme that converts acetoin to 2,3-butanediol, i.e., the adh gene. This resulted in a 2,3-butanediol production rate of 0.002 to 0.007 (g / g bacteria-hour), far greater than the 2,3-butanediol production rate achieved by conventional genetically engineered hydrogen-oxidizing bacteria. While this may vary depending on the environment and reaction conditions, it is possible that the step involving acetolactate decarboxylase, encoded by the alsD gene, is the rate-limiting step in 2,3-butanediol metabolism among the steps involving each metabolic enzyme in the 2,3-butanediol biosynthetic pathway. It has also been found that the expression level of the alsD gene is lower than that of the other two genes, the alsS gene and the adh gene. Therefore, by designing the nucleic acid encoding this alsD gene so that it is located closer to the regulatory sequence, i.e., the arabinose-inducible promoter, the expression level of the alsD gene increases, and the amount of acetolactate decarboxylase increases, which can increase the reaction rate of the reaction in which acetolactate decarboxylase contributes in the step in which acetolactate decarboxylase is involved. Therefore, it is believed that placing the alsD gene closer to the regulatory sequence increased the amount of 2,3-butanediol produced.

[0059] Hydrogen-oxidizing bacteria are known to be species capable of using acetoin as a carbon source. Therefore, hydrogen-oxidizing bacteria can express endogenous acetoin-degrading enzymes. These endogenous acetoin-degrading enzymes oxidatively cleave acetoin into two C2 hydrocarbons. In other words, when acetoin is produced by expression of genes introduced into hydrogen-oxidizing bacteria via transformation, there is a risk that the endogenous acetoin-degrading enzymes of the hydrogen-oxidizing bacteria themselves may degrade acetoin into metabolites other than 2,3-butanediol. The endogenous acetoin-degrading enzymes may react competitively with the expressed acetoin dehydrogenase for acetoin produced by transformation, potentially reducing the amount of 2,3-butanediol produced from acetoin. For this reason, in conventional genetically engineered hydrogen-oxidizing bacteria for 2,3-butanediol production, the acoXABC gene cluster, a group of endogenous enzymes involved in acetoin degradation, is deleted to effectively utilize acetoin as a substrate for the 2,3-butanediol biosynthetic pathway. This reduces or eliminates the activity of endogenous acetoin-degrading enzymes that metabolize acetoin by oxidative cleavage, thereby inhibiting the decomposition of acetoin.

[0060] However, producing such recombinant hydrogen-oxidizing bacteria requires a great deal of effort. The transformant of the present invention can produce significantly high amounts of 2,3-butanediol without deleting the endogenous acoXABC genes. In the 2,3-butanediol-producing transformant of the present invention, the endogenous acoXABC gene cluster may be maintained, or may be partially or entirely deleted, inactivated, or disrupted. Since the present invention does not require a treatment to disrupt the endogenous acoXABC gene cluster, a transformant with excellent 2,3-butanediol production can be obtained more easily.

[0061] 2,3-butanediol can be produced by reacting the transformant of the present invention described above in a reaction solution containing a carbon source. Therefore, the present invention encompasses a method for producing 2,3-butanediol, which comprises culturing such a transformant under conditions for producing 2,3-butanediol and for a sufficient period of time. The carbon source can be used alone or in combination with two or more. Preferably, the carbon source is carbonate ion and / or CO2. The method for producing 2,3-butanediol includes the step of culturing the transformant by introducing CO2, hydrogen, and air (oxygen) into a medium containing the transformant of the present invention. The medium, culture temperature, pH, and other parameters used are not particularly limited as long as the objective is achieved, and can be selected as desired by those skilled in the art depending on factors such as the type of bacteria. Preferably, the growth rate of the transformant is approximately equivalent to that of the hydrogen-oxidizing bacterium before transformation, even after the introduction of enzyme genes involved in 2,3-butanediol production and the disruption of the phaC1AB1 operon involved in PHB synthesis by transformation.

[0062] The method for producing 2,3-butanediol from hydrogen-oxidizing bacteria according to the present invention preferably includes a step of removing cells from the culture filtrate obtained in the culturing step, and a step of isolating 2,3-butanediol from the cell-removed culture filtrate. 2,3-butanediol can be collected from the culture by applying a general method for isolating and purifying ordinary water-soluble neutral substances.

[0063] The present invention also encompasses a composition for producing 2,3-butanediol, which comprises at least one selected from the group consisting of a culture of the transformant, i.e., recombinant hydrogen-oxidizing bacterium, obtained using the transformant according to the present invention, the recombinant hydrogen-oxidizing bacterium, a lysate of the bacterium, and an extract of the culture or the lysate.

[0064] The transformant of the present invention may incorporate a recombinant vector containing a gene of interest for expressing a desired enzyme in the transformant. Introduction (transformation) of a recombinant vector into a host cell can be carried out using known methods. Introduction methods mainly include methods using an expression vector and genome recombination methods using a suicide vector. A known broad-host-range vector can be used as the expression vector used for introduction. Introduction methods may be selected from bacterial protoplast fusion, electroporation, gene gun techniques, and infection with a viral vector. The suicide vector used for introduction is not limited as long as it is replicable in the host cell, but examples include suicide vectors for homologous recombination introduction, particularly pLO3. Homologous recombination can be carried out, for example, by inserting a gene of interest into a sequence homologous to a genomic sequence and then introducing the DNA fragment into the cell by electroporation to induce homologous recombination. An expression vector and a suicide vector may also be combined. Here, a recombinant vector refers to a recombinant nucleic acid molecule capable of transmitting an operably linked polynucleotide of interest.

[0065] Typically, when a mutation is introduced into a gene and the target gene is to be disrupted or knocked out, an appropriate nucleic acid construct or vector can be designed to be integrated into the region of the target gene in the genome of the parent microorganism to disrupt the gene. In the present invention, a polynucleotide encoding a protein involved in 2,3-butanediol production is introduced by genetic recombination techniques to disrupt, for example, the phaC1AB1 operon involved in PHB synthesis in the hydrogen-oxidizing bacterium, and the host hydrogen-oxidizing bacterium is transformed.

[0066] The present invention encompasses vectors for introducing a gene of interest, i.e., a polynucleotide encoding a protein involved in 2,3-butanediol production, into hydrogen-oxidizing bacteria. As described above, the pathway for 2,3-butanediol production is based on three precursors: pyruvate, acetolactate, and acetoin. Therefore, the vector of the present invention is a nucleic acid construct for gene expression, comprising a nucleic acid encoding a gene capable of expressing an enzyme that synthesizes acetolactate from pyruvate, a nucleic acid encoding a gene capable of expressing an enzyme that converts acetolactate to acetoin, and a nucleic acid encoding a gene capable of expressing an enzyme that converts acetoin to 2,3-butanediol. The gene of interest is preferably linked to a promoter for expression control. As described above, the promoter is not particularly limited as long as it functions within the hydrogen-oxidizing bacteria cells into which it is introduced. Furthermore, the vector may also contain polynucleotides necessary for expression of the gene of interest, such as a marker gene for selecting strains into which the gene of interest has been introduced, a terminator, a restriction enzyme recognition site, and polynucleotides necessary for cloning genes related to auxotrophy (e.g., amino acids) and drug resistance genes.

[0067] Specifically, the vector of the present invention comprises a nucleic acid sequence including a gene capable of expressing an enzyme that synthesizes acetolactate from pyruvate, a gene capable of expressing an enzyme that converts acetolactate to acetoin, and a gene capable of expressing an enzyme that converts acetoin to 2,3-butanediol, wherein the nucleic acid sequence is operably linked to a regulatory sequence for expression of three enzymes in the hydrogen-oxidizing bacterium, and the gene capable of expressing the enzyme that converts acetolactate to acetoin is located upstream of the gene capable of expressing the enzyme that synthesizes acetolactate from pyruvate and the gene capable of expressing the enzyme that converts acetoin to 2,3-butanediol. As described above, by transforming a hydrogen-oxidizing bacterium so that the genes are arranged in this order, starting from the position closest to the regulatory sequence and proceeding downstream, a hydrogen-oxidizing bacterium transformant with excellent 2,3-butanediol production ability can be obtained. The three nucleic acids to be introduced, the regulatory sequences, and the promoters contained in the regulatory sequences are as described above for the transformant of the present invention.

[0068] When producing 2,3-butanediol by culturing the transformant of the present invention, the following production conditions may be employed. The transformant of the present invention may be cultured using a heterotrophic medium or an autotrophic medium. Furthermore, a heterotrophic medium and an autotrophic medium may be used in combination at each stage of culture.

[0069] (Preparation of Heterotrophic Medium) Nutrient components used in the heterotrophic medium include Bacto Yeast Extract, tryptone, beef extract, and the like.

[0070] (Preparation of Autotrophic Medium) Components that may be included in the autotrophic medium include nitrogen sources such as ammonia, ammonium (e.g., ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4), ammonium ferric citrate), nitrates (e.g., potassium nitrate (KNO3)), urea, or organic nitrogen sources; phosphates (e.g., sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), phosphoric acid (H3PO4), potassium dithiophosphate (K3PS2O2), potassium orthophosphate, etc. sulfates (e.g., ammonium sulfate ((NH4)2SO4), magnesium sulfate heptahydrate (MgSO4.7H2O)); yeast extract; chelated iron; potassium salts, potassium iodide (KI), potassium bromide (KBr)); and other inorganic salts, minerals, and micronutrients (e.g., sodium chloride (NaCl), or magnesium chloride (MgCl2), calcium chloride (CaCl2) or calcium carbonate (CaCO3), manganese sulfate heptahydrate (MnSO4.7H2O) or or manganese chloride (MnCl2), ferric chloride hexahydrate (FeCl3.6H2O), ferrous sulfate heptahydrate (FeSO4.7H2O), or ferrous chloride tetrahydrate (FeCl2.4H2O), sodium bicarbonate (NaHCO3) or sodium carbonate (Na2CO3), zinc sulfate (ZnSO4) or zinc chloride (ZnCl2), ammonium molybdate (NH4MoO4) or sodium molybdate dihydrate (Na2MoO4.2H2O), cuprous sulfate (CuSO4) or copper chloride dihydrate (CuCl2.2H2O ), cobalt chloride hexahydrate (CoCl2.6H2O), aluminum chloride hexahydrate (AlCl3.6H2O), lithium chloride (LiCl), boric acid (H3BO3), nickel chloride hexahydrate (NiCl2.6H2O), tin chloride monohydrate (SnCl2.H2O), barium chloride dihydrate (BaCl2.2H2O), copper selenate pentahydrate (CuSeO4.5H2O) or one or more inorganic salts of sodium selenite (Na2SeO3), sodium metavanadate (NaVO3), chromium salts).For example, the growth and proliferation of the transformant of the present invention can be promoted by including the above-mentioned micronutrients.

[0071] Suitable inorganic salts that may be included in the autotrophic medium include ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, sodium bicarbonate, ferric ammonium citrate, ferric chloride hexahydrate, ammonium chloride, calcium chloride, nickel chloride hexahydrate, and the like.

[0072] When culturing the transformant of the present invention, it is preferable to carry out the culture by blowing CO2, H2, air or O2 into the culture solution (medium).

[0073] The culture conditions are not particularly limited, and typical hydrogen-oxidizing bacteria culture conditions can be used. For example, the ratio of H2, O2, and CO2 gases blown into the culture medium is preferably H2:O2:CO2 = 4-90:2-20:0.01-30. The supplied H2, O2, and CO2 substrate gases do not need to be 100% pure three-component systems and may contain gases and impurities other than hydrogen, carbon dioxide, and oxygen. For example, the hydrogen concentration in the culture tank should be approximately 4-99 vol%, preferably 30-80 vol%, the oxygen concentration should be 2-20 vol%, preferably 4-10 vol%, and the carbon dioxide concentration should be approximately 0.01-30 vol%, preferably 0.04-10 vol%.

[0074] The above mixed gas can be supplied to the culture medium at a flow rate of 70 to 4000 mL / min. When O2 is introduced as air, the air flow rate should be adjusted so that the O2 flow rate is within the above range. Specifically, CO2 is preferably 10 to 1000 mL / min, H2 is 50 to 2000 mL / min, and O2 is 10 to 1000 mL / min.

[0075] The substrate gas may be hydrogen exhaust gas containing by-product hydrogen and the like emitted from a factory, combustion exhaust gas containing CO and the like emitted from a factory, or air. When CO is collected from exhaust gas, it can be concentrated and used. Concentration methods include the pressure swing adsorption (PSA) method, in which CO is adsorbed onto zeolite or the like under pressure to concentrate it, and then the CO is released by reducing the pressure, and the so-called amine method, in which CO is absorbed into an amine solution to concentrate it, and then the solution is heated to release the CO. Furthermore, H can be reused by separating only H using a gas separation membrane and returning it to the culture tank.

[0076] The temperature during cultivation is not particularly limited and may be set to a temperature suitable for culturing the hydrogen-oxidizing bacteria used, for example, 25 to 35°C. The cultivation time is also not particularly limited, but may be, for example, 30 to 80 hours.

[0077] For example, sodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (KH2PO4), or sodium bicarbonate (NaHCO3) may be added to the culture medium to provide pH buffering capacity.

[0078] For example, after the transformant of the present invention has grown to a certain number, a substance that induces gene expression driven by an inducible promoter is added to the culture medium, and the transformant of the present invention is further cultured. For example, in the case of an arabinose-inducible promoter, arabinose is added.

[0079] After the cultivation is completed, solid-liquid separation is performed by filtration or centrifugation to remove the transformant of the present invention and solid impurities from the culture solution containing 2,3-butanediol. Thereafter, 2,3-butanediol is separated and purified from the culture solution by solvent extraction, distillation, or the like.

[0080] The present invention will be specifically described based on examples, but the present invention is not limited to these.

[0081] (1) Preparation of Homologous Recombination Vector for Examples <Preparation of alsD-alsS-adh Recombination Vector pLO3-1> An artificial gene was prepared by arranging a 750 bp upstream region of the phaC1 gene derived from the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain, an araC gene derived from Escherichia coli, an arabinose-responsive promoter, downstream of which were the alsD gene (SEQ ID NO: 1) derived from Bacillus subtilis, the alsS gene (SEQ ID NO: 2) derived from Bacillus subtilis, and the adh gene (SEQ ID NO: 3) derived from Clostridium beijerinckii, as well as a 750 bp downstream region of the phaB1 gene derived from Cupriavidus necator H16 strain. The constructed artificial gene was integrated into the suicide vector pLO3 (SEQ ID NO: 4) (synthesized using Vector Builder) by seamless cloning. This work was outsourced to Eurofins Genomics. This vector for Example 1 was named pLO3-1. Figure 1 shows the mechanism by which the target gene was integrated into a predetermined location in the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator strain H16 by homologous recombination using pLO3-1 in the production of Example Transformant 1, as well as the structure of the recombinant strain for Example 1 obtained by recombination.

[0082] <alsD * -alsS * -adh *Preparation of recombinant vector pLO3-2> A 750 bp region upstream of the phaC1 gene derived from the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain, the araC gene derived from Escherichia coli, an arabinose-responsive promoter, and downstream thereof, the alsD gene (SEQ ID NO: 14) derived from the Enterobacter kobei ENHKU01 strain, the alsS gene (SEQ ID NO: 15) derived from Acinetobacter venetianus, the adh gene (SEQ ID NO: 16) derived from Acinetobacter lactucae strain OTEC-02, and the adh gene (SEQ ID NO: 17) derived from Cupriavidus necator H16 strain were used. An artificial gene was created by locating a 750-bp region downstream of the phaB1 gene derived from the Cupriavidus necator H16 strain. The constructed artificial gene was seamlessly cloned into the suicide vector pLO3 (SEQ ID NO: 4) (synthesized using Vector Builder). This work was outsourced to Eurofins Genomics. This vector for Example 3 was named pLO3-2. Figure 3 shows the mechanism by which the target gene was integrated into a predetermined location in the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain by homologous recombination using pLO3-2 in the production of Example Transformant 3, as well as the structure of the recombinant strain for Example 3 obtained by recombination. In FIG. 3, the alsD gene derived from the Enterobacter kobei ENHKU01 strain, the alsS gene derived from Acinetobacter venetianus, and the adh gene derived from the Acinetobacter lactucae strain OTEC-02 are respectively designated as alsD and alsS. * , alsS * , adh * It is shown as follows.

[0083] <Preparation of alsD-adh-alsS recombination vector pLO3-3> An artificial gene was prepared by arranging a 750 bp region upstream of the phaC1 gene derived from the hydrogen-oxidizing bacterium Cupriavidus necator strain H16, the araC gene derived from Escherichia coli, an arabinose-responsive promoter, downstream of which were the alsD gene (SEQ ID NO: 1) derived from Bacillus subtilis, the adh gene (SEQ ID NO: 3) derived from Clostridium beijerinckii, and the alsS gene (SEQ ID NO: 2) derived from Bacillus subtilis, and further a 750 bp region downstream of the phaB1 gene derived from Cupriavidus necator strain H16. The constructed artificial gene was integrated into the suicide vector pLO3 (SEQ ID NO: 4) (synthesized using Vector Builder) by seamless cloning. This work was outsourced to Eurofins Genomics. This vector for Example 5 was named pLO3-3. Figure 4 shows the mechanism by which the target gene was integrated into a predetermined position in the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator strain H16 by homologous recombination using pLO3-3 in the production of Example Transformant 5, as well as the structure of the recombinant strain for Example 5 obtained by recombination.

[0084] <alsD * -adh * -alsS *Preparation of recombinant vector pLO3-4> A 750 bp region upstream of the phaC1 gene derived from the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain, the araC gene derived from Escherichia coli, an arabinose-responsive promoter, and downstream thereof, the alsD gene (SEQ ID NO: 14) derived from the Enterobacter kobei ENHKU01 strain, the adh gene (SEQ ID NO: 16) derived from Acinetobacter lactucae strain OTEC-02, the alsS gene (SEQ ID NO: 15) derived from Acinetobacter venetianus, and the alsD gene (SEQ ID NO: 16) derived from Cupriavidus necator H16 strain were used. An artificial gene was created by locating a 750-bp region downstream of the phaB1 gene derived from the Cupriavidus necator H16 strain. The constructed artificial gene was seamlessly cloned into the suicide vector pLO3 (SEQ ID NO: 4) (synthesized using Vector Builder). This work was outsourced to Eurofins Genomics. This vector for Example 6 was named pLO3-4. Figure 5 shows the mechanism by which the target gene was integrated into a predetermined location in the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain by homologous recombination using pLO3-4 in the production of Example Transformant 6, as well as the structure of the recombinant strain for Example 6 obtained by recombination. In FIG. 5, the alsD gene derived from the Enterobacter kobei ENHKU01 strain, the adh gene derived from the Acinetobacter lactucae strain OTEC-02, and the alsS gene derived from Acinetobacter venetianus are respectively designated as alsD. * , adh * , alsS * It is shown as follows.

[0085] Example 1 Preparation of Transformant 1 for Example <Preparation of E. coli S17-1 Competent Cells> (2) The E. coli S17-1 strain was inoculated into 10 mL of LB liquid medium (Miller) (Sm+) and cultured overnight with shaking at 37°C and 140 rpm. The entire culture solution after overnight culture was transferred to 1 L of LB liquid medium (Miller), placed in a 2 L Erlenmeyer flask, and cultured with shaking at 30°C and 140 rpm. When the OD600 reached 0.56, the culture solution was dispensed into 50 mL centrifuge tubes in 40 mL aliquots and centrifuged at 3,350 x g for 15 minutes at 4°C (Kubota Shoji Co., Ltd., 7780II). The supernatant was discarded, and the precipitate was suspended in 30 mL of ice-cold sterilized water and centrifuged at 3,350 x g for 15 minutes at 4°C. The supernatant was discarded again, and the precipitate was suspended in 10 mL of ice-cold sterile water and centrifuged at 3,350 × g for 15 minutes at 4 ° C. The supernatant was discarded, and the precipitate was suspended in 1 mL of 10% glycerol solution and centrifuged at 3,350 × g for 15 minutes at 4 ° C. The supernatant was discarded, and the precipitate was suspended in 1 mL of 10% glycerol solution. This was dispensed in 50 μL aliquots into 1.5 mL Eppendorf tubes to prepare competent cells. The cells were stored at -80 ° C until use.

[0086] Transformation of Escherichia coli S17-1 with pLO3-1 Vector (3) S17-1 competent cells stored at -80°C were thawed on ice, and 1 μL of 10 ng / μL pLO3-1 was added and mixed by pipetting. After mixing, the mixture was immediately placed in an electroporation cuvette (Thermo Fisher Scientific No. 5540 1 mm), and transformation was performed by electroporation using an Eppendorf Electroporator 2510. Electroporation conditions were 2000 V for 5 seconds. The mixture of E. coli and pLO3-1 was then transferred from the cuvette to a 1.5 mL Eppendorf tube, and 1 mL of LB liquid medium (Miller) was added. The mixture was cultured at 37°C and 200 rpm for 1 hour with shaking. After the 1-hour curing, the mixture was centrifuged at 3,350 × g for 5 minutes at room temperature (Kubota Shoji Co., Ltd., 7780II), 900 μL of the supernatant was discarded, and the pellet was suspended in the remaining 100 μL. The resulting suspension was plated on LB agar medium (Miller) (Tc+) and incubated at 30°C for 2 days.

[0087] <Cultivation of E. coli and H16 strain for co-culture> (4) 100 μL of hydrogen-oxidizing bacteria Cupriavidus necator H16 strain was taken from a glycerol stock and inoculated into 10 mL of FN(+) liquid medium in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 3 days. This culture was designated as co-culture sample (A). The E. coli colonies obtained in (3) were attached with the back of a toothpick and inoculated into 10 mL of LB liquid medium (Miller) (Tc+) in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 2 days. This culture was designated as co-culture sample (B).

[0088] <Co-culture> (5) The entire co-culture sample (A) from (4) was transferred to a 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. The entire co-culture sample (B) from (4) was transferred to another 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 20 mL of FN(-) liquid medium. This was again centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. 200 μL of each of the above two suspensions was transferred to a 1.5 mL Eppendorf tube, mixed by pipetting, and then a total of 400 μL was added dropwise to LB agar medium. After addition, the dish was covered with a lid and left in a clean bench for 5 hours to allow the water to evaporate naturally. Thereafter, the petri dish was transferred into an incubator (Yamato Scientific Co., Ltd., IN604W) and cultured overnight at 30°C.

[0089] <Primary Selection> (6) A Pasteur pipette was heated with a gas burner to form a spreader-like shape. The entire paste-like culture obtained in (5) was collected using a spreader-shaped Pasteur pipette and suspended in 10 mL of FN(-) liquid medium placed in a 50 mL centrifuge tube. 50 μL of the suspension was inoculated onto FN(+) (Tc+) agar medium, spread over the entire dish using a spreader, and then placed in an incubator for static culture at 37°C for 3 days.

[0090] <PCR check of primary selection colonies> (7) Several colonies formed in the Petri dish in (6) were selected, and colony PCR was used to determine whether the recombinant gene had been inserted into the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain. KOD Fx Neo (manufactured by Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were phaC1up1138 (SEQ ID NO: 5) and phaCr (SEQ ID NO: 6). The PCR conditions were 94°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds and 68°C for 3 minutes. An Applied Biosystems 2720 thermal cycler was used for PCR. The resulting PCR products were electrophoresed on a 1% agarose gel at 100 V for 25 minutes, stained, and then judged whether or not a PCR product of the desired length had been obtained (Mupid (registered trademark)-exU, manufactured by ADV).

[0091] <Secondary Selection> (8) Colonies from which PCR products of the desired length were obtained in (7) were picked with an autoclave-sterilized toothpick and inoculated into 10 mL of LB sucrose liquid medium in a 50 mL centrifuge tube and cultured overnight. The entire culture was centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 200 μL of LB liquid medium (Miller), followed by a 100,000-fold dilution using LB liquid medium (Miller). 50 μL of this suspension was seeded on LB sucrose agar medium, spread over the entire dish with a spreader, and then placed in an incubator for static culture at 30°C for 3 days.

[0092] <PCR check of secondary selection colonies> (9) Several colonies formed in the Petri dish (8) were selected, and colony PCR was used to determine whether the vector portion of the suicide vector pLO3 had disappeared. KOD Fx Neo (manufactured by Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were a combination of phaC1up1138 (SEQ ID NO: 5) and araB (SEQ ID NO: 7), a combination of sadh for (SEQ ID NO: 8) and phaB1downrv5 (SEQ ID NO: 9), or a combination of adh2 for (SEQ ID NO: 13) and phaB1downrv5 (SEQ ID NO: 9). The PCR conditions were 94 ° C for 2 minutes, followed by 35 cycles of 98 ° C for 10 seconds and 68 ° C for 3 minutes. The resulting PCR product was electrophoresed on a 1% agarose gel at 100 V for 25 minutes, and after staining, it was determined whether a PCR product of the desired length had been obtained. The colony from which a PCR product of the desired length was obtained was a strain in which the phaC1AB1 gene cluster originally present in the genome had been replaced with the artificial gene prepared in (1). This was designated as the genetically modified strain for Example 1, Example Transformant 1.

[0093] Example 3 Preparation of Example Transformant 3 The genetically modified strain for Example 3, Example Transformant 3, was obtained in the same manner as in (2) to (9) above, except that the transformation of E. coli S17-1 in (3) was carried out using the pLO3-2 vector.

[0094] Example 5 Preparation of Example Transformant 5

[0123] The above steps (2) to (9) were repeated except that the transformation of Escherichia coli S17-1 in (3) was carried out using the pLO3-3 vector, to obtain Example Transformant 5, a recombinant strain for Example 5.

[0095] Example 6 Preparation of Example Transformant 6 The genetically modified strain for Example 6, Example Transformant 6, was obtained in the same manner as in (2) to (9) above, except that the transformation of Escherichia coli S17-1 in (3) was carried out using the pLO3-4 vector.

[0096] Example 2: Preparation of Example Transformant 2 <Preparation of acoXABC knockout vector> (10) To delete the acoXABC gene in the genomic DNA of the genetically modified strains used in Examples 1 and 3, a DNA fragment containing 1,024 bp upstream of the acoX gene followed by 821 bp downstream of the acoC gene was prepared using an artificial gene synthesis service (Eurofins Genomics, Inc.). This DNA fragment was integrated into the suicide vector pLO3 used in (1) by seamless PCR. The resulting vector was designated the acoXABC gene deletion vector pLO3ΔacoXABC. Figure 1 shows the mechanism of homologous recombination using pLO3ΔacoXABC in the preparation of Example Transformant 2 and the structure of the recombinant strain used in Example 2 obtained by recombination.

[0097] Transformation of Escherichia coli S17-1 with pLO3ΔacoXABC Vector (11) The S17-1 competent cells stored at -80°C in (2) were thawed on ice, and 1 μL of 10 ng / μL pLO3ΔacoXABC was added and mixed by pipetting. After mixing, the mixture was immediately placed in an electroporation cuvette (Thermo Fisher Scientific No. 5540 1 mm), and transformation was performed by electroporation using an Eppendorf Electroporator 2510. Electroporation conditions were 2000 V for 5 seconds. The mixture of E. coli and pLO3ΔacoXABC was then transferred from the cuvette to a 1.5 mL Eppendorf tube, and 1 mL of LB liquid medium (Miller) was added. The mixture was then cultured at 37°C with shaking at 200 rpm for 1 hour. After the 1-hour curing, the mixture was centrifuged at 5,000 rpm for 5 minutes at room temperature, 900 μL of the supernatant was discarded, and the pellet was suspended in the remaining 100 μL. The resulting suspension was inoculated onto LB agar medium (Miller) (Tc+) and incubated at 30°C for 2 days.

[0098] <Cultivation of E. coli for co-culture and the genetically modified strain for Example 1> (12) The genetically modified strain for Example 1 obtained in (9) was attached with the back of an autoclave-sterilized toothpick and inoculated into 10 mL of FN(+) liquid medium in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 3 days. This culture solution was designated as co-culture sample (C). The E. coli colony obtained in (11) was attached with the back of a toothpick and inoculated into 10 mL of LB liquid medium (Miller) (Tc+) in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 2 days. This culture solution was designated as co-culture sample (D).

[0099] <Co-culture> (13) The entire co-culture sample (C) from (12) was transferred to a 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. The entire co-culture sample (D) from (12) was transferred to another 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 20 mL of FN(-) liquid medium. This was again centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. 200 μL of each of the above two suspensions was transferred to a 1.5 mL Eppendorf tube, mixed by pipetting, and then a total of 400 μL was added dropwise to LB agar medium. After addition, the dish was covered with a lid and left in a clean bench for 5 hours to allow the water to evaporate naturally. Thereafter, the petri dish was transferred into an incubator and cultured overnight at 30°C.

[0100] <Primary Selection> (14) A Pasteur pipette was heated with a gas burner to form a spreader-like shape. The entire paste-like culture obtained in (13) was collected using a spreader-shaped Pasteur pipette and suspended in 10 mL of FN(-) liquid medium placed in a 50 mL centrifuge tube. 50 μL of the suspension was inoculated onto FN(+) (Tc+) agar medium, spread over the entire dish using a spreader, and then placed in an incubator for static culture at 37°C for 3 days.

[0101] <PCR check of primary selection colonies> (15) Several colonies formed in the petri dish (14) were selected, and colony PCR was used to determine whether the recombinant gene had been inserted into the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain. KOD Fx Neo (Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were acoXUPupF (SEQ ID NO: 10) and acoCdnR (SEQ ID NO: 11). The PCR conditions were 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 3 minutes (35 cycles). The resulting PCR product was electrophoresed on a 1% agarose gel at 100V for 25 minutes, stained, and then evaluated for the desired length of the PCR product.

[0102] <Secondary Selection> (16) Colonies that yielded PCR products of the desired length in (15) were picked with an autoclave-sterilized toothpick and inoculated into 10 mL of LB sucrose liquid medium in a 50 mL centrifuge tube and cultured overnight. The entire culture was centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 200 μL of LB liquid medium (Miller), followed by a 100,000-fold dilution using LB liquid medium (Miller). 50 μL of this suspension was plated on LB sucrose agar medium, spread using a spreader, and then placed in an incubator for static culture at 30°C for 3 days.

[0103] <PCR check of secondary selection colonies> (17) Several colonies formed in the Petri dish (16) were selected, and colony PCR was used to determine whether the vector portion of the suicide vector pLO3 had disappeared. KOD Fx Neo (manufactured by Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were acoXUPupF (SEQ ID NO: 10) and acoCDndnR (SEQ ID NO: 12). The PCR conditions were 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 3 minutes, for a total of 35 cycles. The resulting PCR product was electrophoresed on a 1% agarose gel at 100V for 25 minutes, and stained to determine whether a PCR product of the desired length had been obtained. Colonies that yielded PCR products of the desired length were strains lacking the acoXABC gene cluster originally present in the genome. This was designated the genetically modified strain for Example 2, Example Transformant 2.

[0104] Example 4 Preparation of Example Transformant 4 The above steps (10) to (17) were repeated to obtain the genetically modified strain for Example 4, Example Transformant 4, except that the co-culture sample (C) in (12) was prepared using the genetically modified strain for Example 3. Note that Figure 3 shows the mechanism of homologous recombination using pLO3ΔacoXABC in the production of Example Transformant 4, and the structure of the recombinant strain for Example 4 obtained by recombination.

[0105] Comparative Example 1 Preparation of Comparative Transformant 1 <Preparation of alsS-alsD-adh Recombination Vector pLO3-0> (18) An artificial gene was prepared by arranging a 750 bp upstream region of the phaC1 gene derived from the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain, an araC gene derived from Escherichia coli, an arabinose-responsive promoter, downstream of which were the alsS gene derived from Bacillus subtilis, the alsD gene derived from Bacillus subtilis, and the adh gene derived from Clostridium beijerinckii, as well as a 750 bp downstream region of the phaB1 gene derived from Cupriavidus necator H16 strain. The constructed artificial gene was integrated into the suicide vector pLO3 (synthesized using Vector Builder) by seamless cloning. This work was outsourced to Eurofins Genomics. This vector for comparative purposes was named pLO3-0. Figure 2 shows the mechanism by which the target gene was integrated into a predetermined location in the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator strain H16 by homologous recombination using pLO3-0 in the production of comparative transformant 1, as well as the structure of the recombinant strain obtained by recombination.

[0106] <Preparation of Escherichia coli S17-1 Competent Cells> (19) Competent cells of Escherichia coli S17-1 were prepared in the same manner as in (2).

[0107] Transformation of Escherichia coli S17-1 with pLO3-0 Vector (20) S17-1 competent cells stored at -80°C were thawed on ice, and 1 μL of 10 ng / μL pLO3-0 was added and mixed by pipetting. After mixing, the mixture was immediately placed in an electroporation cuvette (Thermo Fisher Scientific No. 5540 1 mm), and transformation was performed by electroporation using an Eppendorf Electroporator 2510. Electroporation conditions were 2000 V for 5 seconds. The mixture of E. coli and pLO3-0 was then transferred from the cuvette to a 1.5 mL Eppendorf tube, and 1 mL of LB liquid medium (Miller) was added. The mixture was then cultured at 37°C and 200 rpm for 1 hour with shaking. After the 1-hour curing, the mixture was centrifuged at 3,350 × g for 5 minutes at room temperature, 900 μL of the supernatant was discarded, and the pellet was suspended in the remaining 100 μL. The resulting suspension was inoculated onto LB agar medium (Miller) (Tc+) and incubated at 30°C for 2 days.

[0108] <Cultivation of Escherichia coli and H16 strain for co-culture> (21) 100 μL of hydrogen-oxidizing bacteria Cupriavidus necator H16 strain was taken from a glycerol stock and inoculated into 10 mL of FN(+) liquid medium in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 3 days. This culture was designated as co-culture sample (E). The Escherichia coli colony obtained in (20) was attached with the back of a toothpick and inoculated into 10 mL of LB liquid medium (Miller) (Tc+) in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 2 days. This culture was designated as co-culture sample (F).

[0109] <Co-culture> (22) The entire co-culture sample (E) from (21) was transferred to a 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. The entire co-culture sample (F) from (21) was transferred to another 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 20 mL of FN(-) liquid medium. This was again centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. 200 μL of each of the above two suspensions was transferred to a 1.5 mL Eppendorf tube, mixed by pipetting, and then a total of 400 μL was added dropwise to LB agar medium. After addition, the dish was covered with a lid and left in a clean bench for 5 hours to allow natural evaporation of the water. Thereafter, the petri dish was transferred into an incubator and cultured overnight at 30°C.

[0110] <Primary Selection> (23) A Pasteur pipette was heated with a gas burner to form a spreader-like shape. The entire paste-like culture obtained in (22) was collected using the spreader-shaped Pasteur pipette and suspended in 10 mL of FN(-) liquid medium placed in a 50 mL centrifuge tube. 50 μL of the suspension was inoculated onto FN(+) (Tc+) agar medium, spread over the entire surface of the dish using a spreader, and then placed in an incubator for static culture at 37°C for 3 days.

[0111] <PCR check of primary selection colonies> (24) Several colonies formed in the Petri dish (23) were selected, and colony PCR was used to determine whether the recombinant gene had been inserted into the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain. KOD Fx Neo (Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were phaC1up1138 (SEQ ID NO: 5) and phaCr (SEQ ID NO: 6). The PCR conditions were 94°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds and 68°C for 3 minutes. The resulting PCR product was electrophoresed on a 1% agarose gel at 100V for 25 minutes, and after staining, it was determined whether a PCR product of the desired length had been obtained.

[0112] <Secondary Selection> (25) Colonies that yielded PCR products of the desired length in (24) were picked with an autoclave-sterilized toothpick and inoculated into 10 mL of LB sucrose liquid medium in a 50 mL centrifuge tube and cultured overnight. The entire culture was centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 200 μL of LB liquid medium (Miller), followed by a 100,000-fold dilution using LB liquid medium (Miller). 50 μL of this suspension was seeded on LB sucrose agar medium, spread over the entire dish using a spreader, and then placed in an incubator for static culture at 30°C for 3 days.

[0113] <PCR check of secondary selection colonies> (26) Several colonies formed in the Petri dish (25) were selected, and colony PCR was used to determine whether the vector portion of the suicide vector pLO3 had disappeared. KOD Fx Neo (manufactured by Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were a combination of phaC1up1138 (SEQ ID NO: 5) and araB (SEQ ID NO: 7), and a combination of sadh for (SEQ ID NO: 8) and phaB1downrv5 (SEQ ID NO: 9). The PCR conditions were 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 3 minutes, for a total of 35 cycles. The resulting PCR product was electrophoresed on a 1% agarose gel at 100V for 25 minutes, and after staining, it was determined whether a PCR product of the desired length had been obtained. The colony from which a PCR product of the desired length was obtained was a strain in which the phaC1AB1 gene cluster originally present in the genome had been replaced with the artificial gene prepared in (18). This was designated as the genetically modified strain for Comparative Example 1, Comparative Example Transformant 1.

[0114] Comparative Example 2 Preparation of Comparative Transformant 2 <Transformation of Escherichia coli S17-1 with pLO3ΔacoXABC Vector> (27) In the same manner as in (11), S17-1 competent cells were transformed with the acoXABC gene deletion vector pLO3ΔacoXABC prepared in (10).

[0115] <Cultivation of E. coli for co-culture and the genetically modified strain for Comparative Example 1> (28) The genetically modified strain for Comparative Example 1 obtained in (26) was attached to the sample with the back of an autoclave-sterilized toothpick and inoculated into 10 mL of FN(+) liquid medium in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 3 days. This culture solution was designated as co-culture sample (G). The E. coli colony obtained in (27) was attached to the sample with the back of a toothpick and inoculated into 10 mL of LB liquid medium (Miller) (Tc+) in a 50 mL centrifuge tube. This was cultured with shaking at 30°C and 140 rpm for 2 days. This culture solution was designated as co-culture sample (H).

[0116] <Co-culture> The entire volume of co-culture sample (G) from (29) (28) was transferred to a 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. The entire volume of co-culture sample (H) from (28) was transferred to another 50 mL centrifuge tube. It was then centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 20 mL of FN(-) liquid medium. This was again centrifuged at 5,000 rpm for 5 minutes at room temperature and suspended in 600 μL of FN(-) liquid medium. 200 μL of each of the above two suspensions was transferred to a 1.5 mL Eppendorf tube, mixed by pipetting, and then a total volume of 400 μL was added dropwise to LB agar medium. After addition, the dish was covered with a lid and left in a clean bench for 5 hours to allow natural evaporation of water. Thereafter, the petri dish was transferred into an incubator and cultured overnight at 30°C.

[0117] <Primary Selection> (30) A Pasteur pipette was heated with a gas burner to form a spreader-like shape. The entire paste-like culture obtained in (29) was collected using a spreader-shaped Pasteur pipette and suspended in 10 mL of FN(-) liquid medium placed in a 50 mL centrifuge tube. 50 μL of the suspension was inoculated onto FN(+) (Tc+) agar medium, spread over the entire dish using a spreader, and then placed in an incubator for static culture at 37°C for 3 days.

[0118] <PCR check of primary selection colonies> (31) Several colonies formed in the Petri dish (30) were selected, and colony PCR was used to determine whether the recombinant gene had been inserted into the genomic DNA of the hydrogen-oxidizing bacterium Cupriavidus necator H16 strain. KOD Fx Neo (Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were acoXUPupF (SEQ ID NO: 10) and acoCdnR (SEQ ID NO: 11). The PCR conditions were 94°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds and 68°C for 3 minutes. The resulting PCR product was electrophoresed on a 1% agarose gel at 100V for 25 minutes, and after staining, it was determined whether a PCR product of the desired length had been obtained.

[0119] <Secondary Selection> (32) Colonies that yielded PCR products of the desired length in (31) were picked with an autoclave-sterilized toothpick and inoculated into 10 mL of LB sucrose liquid medium in a 50 mL centrifuge tube and cultured overnight. The entire culture was centrifuged at 3,350 × g for 5 minutes at room temperature. The supernatant was discarded, and the precipitate was suspended in 200 μL of LB liquid medium (Miller), followed by a 100,000-fold dilution using LB liquid medium (Miller). 50 μL of this suspension was plated on LB sucrose agar medium, spread using a spreader, and then placed in an incubator for static culture at 30°C for 3 days.

[0120] <PCR check of secondary selection colonies> (33) Several colonies formed in the Petri dish (32) were selected, and colony PCR was used to determine whether the vector portion of the suicide vector pLO3 had disappeared. KOD Fx Neo (Toyobo Co., Ltd.) was used as the colony PCR reagent. The primers used for PCR were acoXUPupF (SEQ ID NO: 10) and acoCDndnR (SEQ ID NO: 12). The PCR conditions were 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 3 minutes (35 cycles). The resulting PCR product was electrophoresed on a 1% agarose gel at 100V for 25 minutes, stained, and then determined whether a PCR product of the desired length was obtained. Colonies that yielded PCR products of the desired length were strains lacking the acoXABC gene cluster originally present in the genome. This was designated the genetically modified strain for Comparative Example 2, Comparative Example Transformant 2.

[0121] 2,3-Butanediol Production Experiment Using Example Transformants 1 to 6 and Comparative Example Transformants 1 and 2 <Pre-culture under Heterotrophic Conditions> (34) Each colony of the genetically modified strains for Examples 1 to 6 prepared in (9) and (17), respectively, and each colony of the genetically modified strains for Comparative Examples 1 and 2 prepared in (26) and (33), respectively, was attached with the back of a toothpick and inoculated into FN(+) liquid medium placed in a 100 mL Erlenmeyer flask. This was cultured with shaking at 30°C and 140 rpm for 2 days in an incubator (Yamato Scientific Co., Ltd., IN604W).

[0122] <Main culture under autotrophic conditions> (35) 700 mL of FN(-) liquid medium was placed in a homemade incubator made using a 1 L cylindrical container, and the culture solution cultivated in (34) was added so that the OD600nm was 0.05 (Shimadzu Corporation, Biospec-mini). In addition, three types of gases were constantly supplied through a sterilizing filter (Toyo Roshi Kaisha, DISMIC (registered trademark)-25CS) and a kerami filter (AS ONE Corporation, 2-554-01, pore size 40-50 μm). The gases and their supply rates were as follows: hydrogen gas 80 mL / min, carbon dioxide gas 30 mL / min, and air 100 mL / min. The temperature of the culture solution during cultivation was maintained at 28-32°C by controlling a belt heater (Hakko Electric Co., Ltd., SBH2127) with a temperature controller (KETOTEK, KT3100). The solution was stirred at 500±20 rpm with a screw-type stirring blade (Kenis Co., Ltd., four-blade stirring propeller, 65 mm) attached to a motor (Wosune, xd-37Gb555) attached to the top of the incubator.

[0123] <Induction by arabinose> (36) Eight types of genetically modified bacteria were grown, and the OD 600 When the rate of increase became less than 100%, an L(+)-arabinose solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 0.1%.

[0124] <Sampling of Culture Solution for Evaluation> (37) 36 to 48 hours after the induction of expression of the 2,3-butanediol synthesis-related enzymes by arabinose, the culture solution was collected from the culture vessel and used as a sample for evaluating the amount of 2,3-butanediol produced.

[0125] <Solid-liquid separation of sample> (38) The culture medium recovered in (37) was dispensed into 50 mL centrifuge tubes in 40-50 mL aliquots. Then, the mixture was centrifuged at 10,000 × g for 10 minutes using a high-speed centrifuge (7780II, manufactured by Kubota Shoji Co., Ltd.). After centrifugation, the supernatant was transferred to another 50 mL centrifuge tube for measuring 2,3-butanediol, and the precipitate was temporarily stored at −20°C for measuring the dry cell weight.

[0126] <Measurement of dry cell weight> (39) The precipitate obtained in (38) was frozen at −20° C. for 12 hours or more, and then dried in a freeze dryer (Tokyo Rikakikai Co., Ltd., FDU-1200) for 12 hours or more. The weight of the precipitate was then measured using a precision balance (Shimadzu Corporation, AYU120) to obtain the dry cell weight.

[0127] <Pretreatment for measuring the concentration of 2,3-butanediol> (40) The supernatant obtained in (38) was filtered through a syringe filter with a pore size of 0.45 μm (DISMIC (registered trademark)-25CS, manufactured by Toyo Roshi Kaisha, Ltd.) and appropriately diluted with methanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0128] <Measurement of 2,3-butanediol Concentration> (41) Using a GC-MS apparatus (Shimadzu Corporation, GCMS-QP2010, Plus) and a column (Agilent Technologies, Inc., DB-WAX, 30 m × 0.25 mm, id × 0.25 μm, film thickness), the concentration of 2,3-butanediol was measured under the following conditions: Column oven temperature: 40°C (3 min) -10°C / min -250°C (10 min), Carrier gas: He 1.0 mL / min (constant flow mode), Inlet: 250°C, Split: 10:1, Detector: MS, Ionization method: EI method, Electron energy: 70 eV, Interface temperature: 150°C, Ion source temperature: 230°C, Mass range: m / z = 43, 45, 57 (SIM).

[0129] A calibration curve was prepared using 2,3-butanediol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain the 2,3-butanediol concentration of the sample.

[0130] <2,3-Butanediol-producing ability of each transformant> (42) The 2,3-butanediol-producing ability of each of the genetically modified strains used in Examples 1 to 6 and the genetically modified strains used in Comparative Examples 1 and 2 was calculated from the obtained 2,3-butanediol concentration, cell weight, and culture time. The results are shown in Table 1 below.

[0131]

[0132] As shown in Table 1, the transformants of Examples 1 to 4 transformed with the alsD-alsS-adh recombination vector and Examples 5 and 6 transformed with the alsD-adh-alsS recombination vector had 1.5 times or more the ability to produce 2,3-butanediol compared to the transformant of Comparative Example 1 transformed with the alsS-alsD-adh recombination vector or the transformant of Comparative Example 2, which corresponds to a conventional genetically engineered hydrogen-oxidizing bacterium transformed with the alsS-alsD-adh recombination vector and further lacking the endogenous acoXABC gene cluster involved in endogenous acetoin decomposition enzymes. These results indicate that, to increase 2,3-butanediol production, it is preferable to express the three genes involved in the 2,3-butanediol biosynthetic pathway, alsS, alsD, and adh, in the position closest to the regulatory sequence downstream, rather than in the same order as the steps involved in 2,3-butanediol biosynthesis (alsS-alsD-adh). Furthermore, in a strain in which adh was placed after alsD (alsD-adh-alsS), 2,3-butanediol production was reduced to about one-third of that in a strain in which alsS was placed after alsD (alsD-alsS-adh). This suggests that the enzymes play a major role in 2,3-butanediol production in the order alsD, alsS, and adh.

[0133] Furthermore, the 2,3-butanediol production ability of the transformant of Example 1, which retained the endogenous acoXABC gene cluster, was approximately the same as that of the transformant of Example 2, in which the endogenous acoXABC gene cluster was deleted. Similarly, the 2,3-butanediol production ability of the transformant of Example 3, which retained the endogenous acoXABC gene cluster, was also approximately the same as that of the transformant of Example 4, in which the endogenous acoXABC gene cluster was deleted. From these results, it can be understood that in a transformant in which the three genes involved in the 2,3-butanediol biosynthetic pathway, alsS, alsD, and adh, are arranged in the order alsD-alsS-adh from a position closest to the regulatory sequence downstream, expression of endogenous acetoinolytic enzymes during 2,3-butanediol production does not affect the amount of 2,3-butanediol produced. On the other hand, in the transformant of Comparative Example 2 in which the genes encoding the metabolic enzymes involved in the 2,3-butanediol biosynthetic pathway were arranged in the same order as the steps involved in 2,3-butanediol biosynthesis (alsS-alsD-adh) and which retained the endogenous acoXABC gene cluster, almost no production of 2,3-butanediol was observed.

Claims

1. A transformant of a hydrogen-oxidizing bacterium that grows in the presence of oxygen, into which the following have been introduced: (a) a nucleic acid encoding an enzyme that synthesizes acetolactate from pyruvate; (b) a nucleic acid encoding an enzyme that converts acetolactate to acetoin; (c) a nucleic acid encoding an enzyme that converts acetoin to 2,3-butanediol; and (d) a regulatory sequence operably linked to the nucleic acids (a) to (c), wherein the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c), and which is used for producing 2,3-butanediol.

2. The transformant according to claim 1, wherein the hydrogen-oxidizing bacterium is capable of synthesizing pyruvic acid using carbonate ions and / or CO2 as a carbon source.

3. The transformant according to claim 1, wherein the hydrogen-oxidizing bacteria is at least one species selected from the group consisting of hydrogen-oxidizing bacteria belonging to the genus Cupriavidus, hydrogen-oxidizing bacteria belonging to the genus Hydrogenophilus, hydrogen-oxidizing bacteria belonging to the genus Hydrogenobacter, and hydrogen-oxidizing bacteria belonging to the genus Hydrogenovibrio.

4. The transformant according to claim 1, wherein the hydrogen-oxidizing bacterium is a hydrogen-oxidizing bacterium belonging to the genus Cupriavidus or a hydrogen-oxidizing bacterium belonging to the genus Hydrogenophilus.

5. The transformant according to claim 1, wherein the nucleic acid (b) is located upstream of the nucleic acid (a) and the nucleic acid (c), and the nucleic acid (a) is located upstream of the nucleic acid (c).

6. The transformant of claim 1, wherein said regulatory sequence comprises an inducible promoter.

7. The transformant according to claim 1, wherein the introduction of the regulatory sequence and the nucleic acids (a) to (c) removes at least a portion of the nucleic acid region encoding the enzymes that synthesize hydroxyalkanoic acid in the genomic DNA.

8. The transformant according to claim 1, wherein the nucleic acid (a) is derived from a microorganism belonging to the genus Bacillus or Acinetobacter.

9. The transformant according to claim 1, wherein the nucleic acid (b) is derived from a microorganism belonging to the genus Bacillus or Enterobacter.

10. The transformant according to claim 1, wherein the nucleic acid (c) is derived from a microorganism belonging to the genus Clostridium or Acinetobacter.

11. A transformant as described in claim 1, wherein the nucleic acid (b) comprises a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or 14, or a nucleic acid encoding a protein having at least 80% identity to the base sequence shown in SEQ ID NO: 1 or 14 and having catalytic activity for producing acetoin from acetolactate.

12. The transformant according to claim 1, wherein the nucleic acid (a) comprises a nucleic acid comprising the base sequence shown in SEQ ID NO: 2 or 15, or a nucleic acid encoding a protein having at least 80% identity with the base sequence shown in SEQ ID NO: 2 or 15 and having catalytic activity for producing acetolactate from pyruvate.

13. The transformant according to claim 1, wherein the nucleic acid (c) comprises a nucleic acid comprising the base sequence shown in SEQ ID NO: 3 or 16, or a nucleic acid which has at least 80% identity with the base sequence shown in SEQ ID NO: 3 or 16 and encodes a protein having catalytic activity for producing 2,3-butanediol from acetoin.

14. The transformant according to claim 1, wherein the hydrogen-oxidizing bacterium is Cupriavidus necator.

15. The transformant of claim 1, which retains the endogenous acoXABC gene cluster.

16. The transformant according to claim 1, wherein the acoXABC gene cluster in the hydrogen-oxidizing bacterium is at least partially deleted or inactivated.

17. The transformant according to claim 1, wherein the acoXABC gene cluster in said hydrogen-oxidizing bacterium is deleted.

18. A vector comprising a nucleic acid sequence including a gene capable of expressing an enzyme that synthesizes acetolactate from pyruvate, a gene capable of expressing an enzyme that converts acetolactate to acetoin, and a gene capable of expressing an enzyme that converts acetoin to 2,3-butanediol, wherein the nucleic acid sequence is operably linked to a regulatory sequence for expression of the three enzymes in a hydrogen-oxidizing bacterium, and wherein in the nucleic acid sequence, the gene capable of expressing the enzyme that converts acetolactate to acetoin is located upstream of the gene capable of expressing the enzyme that synthesizes acetolactate from pyruvate and the gene capable of expressing the enzyme that converts acetoin to 2,3-butanediol.

19. A method for transformation, comprising transforming hydrogen-oxidizing bacteria with the vector according to claim 18.

20. A method for producing 2,3-butanediol, comprising the step of culturing the transformant according to claim 1.

21. The method for producing 2,3-butanediol according to claim 20, wherein the culturing is carried out by introducing CO2, hydrogen, and oxygen into a medium containing the transformant.

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