Transformant and method for producing hydrogen using the same

Abstract

To provide: a transformant exhibiting enhanced hydrogen productivity; and a method for efficiently producing hydrogen using the transformant.SOLUTION: The present disclosure provides a transformant for producing hydrogen, obtained by introducing following genes (A), (B), and (C) into a microbial host in an expressible manner, where: (A) a gene encoding an electron-bifurcating [FeFe]-hydrogenase or a homolog thereof; (B) a gene encoding an [FeFe]-hydrogenase maturation factor or a homolog thereof; and (C) a gene encoding a ferredoxin or a homolog thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a hydrogen production technology. In one aspect, the present disclosure relates to a transformant that has been subjected to specific genetic manipulation, and a hydrogen production technology using the transformant. [Background technology]

[0002] Hydrogen has attracted attention as a clean energy source that, unlike fossil fuels, does not produce carbon dioxide when burned. For example, when supplied to a fuel cell, hydrogen can be converted into electrical energy with high efficiency. Attempts have been made to develop technologies for producing hydrogen from sugars by microbial fermentation (e.g., Patent Documents 1 and 2), but there is a demand for the creation of microorganisms (transformants) that produce hydrogen more efficiently. For example, Patent Document 3 proposes a recombinant Escherichia coli that has been modified to contain a heterologous gene encoding HydA, a ferredoxin-dependent hydrogenase derived from Clostridium acetobutylicum, heterologous genes encoding HydE, HydF, and HydG, and a heterologous gene encoding the oxidoreductase NFO (NAD(P)H:ferredoxin-oxidoreductase) or GAPOR (glyceraldehyde-3-phosphate:ferredoxin-oxidoreductase). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2006 / 062130 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-209269 [Patent Document 3] Special Publication No. 2010-500001 Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure provides a transformant capable of efficiently producing hydrogen, and a method for producing hydrogen using the transformant. [Means for solving the problem]

[0005] In one aspect, the present disclosure relates to a transformant for producing hydrogen, which is obtained by expressibly introducing the following genes (A), (B), and (C) into a microbial host: (A) a gene encoding an electron-branching [FeFe]-hydrogenase or its homolog; (B) a gene encoding a [FeFe]-hydrogenase maturation factor or its homolog; (C) A gene encoding ferredoxin or its homologue.

[0006] In another aspect, the present disclosure relates to a transformant for producing hydrogen, which is obtained by expressibly introducing gene (a1), gene (b1), gene (c1), gene (d1), gene (e1), gene (f1), and (D) a gene encoding pyruvate ferredoxin oxidoreductase into a microbial host. (a1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; (b1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (c1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3; (d1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 7; (e1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 8; (f1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 9.

[0007] In another aspect, the present disclosure provides a transformant for producing hydrogen, which is obtained by expressibly introducing gene (a2), gene (b2), gene (c2), gene (d2), gene (e2), gene (f2), and (D) a gene encoding pyruvate ferredoxin oxidoreductase into a microbial host, wherein the transformant comprises: The transformant is characterized in that the polypeptides encoded by genes (a2), (b2), and (c2) can form trimers and have electron-branched [FeFe]-hydrogenase activity, and the polypeptides encoded by genes (d2), (e2), and (f2) can have the function of maturing [FeFe]-hydrogenase. (a2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1; (b2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2; (c2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3; (d2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 7; (e2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 8; (f2) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:9.

[0008] In another aspect, the present disclosure relates to a method for producing hydrogen, comprising the step of culturing the transformant of the present disclosure in a reaction solution containing sugars to produce hydrogen. [Effects of the Invention]

[0009] According to one aspect, the present disclosure can provide a transformant capable of efficiently producing hydrogen, and a method for producing hydrogen using the transformant. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a metabolic pathway diagram illustrating the hydrogen biosynthesis pathway in one embodiment of the transformant of the present disclosure, although stoichiometry is not taken into consideration. DETAILED DESCRIPTION OF THE INVENTION

[0011] To improve productivity in fermentative hydrogen production, it is necessary to efficiently utilize NADH in the hydrogen production reaction. However, the hydrogen production reaction from NADH is thermodynamically unfavorable, which creates a bottleneck by slowing down the reaction. On the other hand, although the hydrogen production reaction from reduced ferredoxin is thermodynamically favorable, the ferredoxin-dependent hydrogenase that catalyzes this reaction cannot utilize NADH. Therefore, it was necessary to modify the NADH utilization pathway by introducing a gene encoding an oxidoreductase such as NADH·ferredoxin oxidoreductase.

[0012] In the course of extensive research, the present inventors discovered that a transformant obtained by expressibly introducing a gene encoding an electron-forking [FeFe]-hydrogenase homolog and a gene encoding an [FeFe]-hydrogenase maturation factor homolog into Escherichia coli can produce hydrogen through the function of these introduced genes. In the course of further investigation, the present inventors discovered that a transformant obtained by expressibly introducing a gene encoding a trimeric electron-forking [FeFe]-hydrogenase homolog (EbhABC) and a gene encoding an [FeFe]-hydrogenase maturation factor homolog (HydEFG) into Escherichia coli can be further introduced with a gene encoding a ferredoxin or a homolog thereof, or a gene encoding a pyruvate ferredoxin oxidoreductase, and that this transformant can significantly improve the amount of hydrogen produced from sugars. The present disclosure is based on these discoveries.

[0013] The hydrogenase used to construct the transformant of the present disclosure is an "electron-branching hydrogenase" that drives the hydrogen production reaction from NADH by utilizing the reducing power of ferredoxin. Therefore, hydrogen can be produced using the reducing power of NADH without the need for a separate gene encoding an oxidoreductase. Furthermore, by introducing genes encoding pyruvate:ferredoxin oxidoreductase and / or ferredoxin into the transformant of the present disclosure in an expressible manner together with the hydrogenase to enhance the supply of reduced ferredoxin, the transformant can produce hydrogen more efficiently by utilizing the reducing power of both NADH and ferredoxin. Furthermore, in the transformant into which the above genes have been introduced, enhancing the supply of NADH by disrupting competing pathways that utilize NADH (e.g., the lactate production pathway, the succinate production pathway, the ethanol production pathway, etc.) can produce hydrogen even more efficiently.

[0014] Furthermore, the present inventors have succeeded in expressing a trimeric enzyme as an electron-branching hydrogenase, and have confirmed that this enables more efficient hydrogen production than enzymes with more than three subunits (e.g., tetramers).

[0015] In the present disclosure, the term "transformant obtained by introducing a gene" may include a transformant obtained by introducing the gene, and a transformant that can be obtained by introducing the gene.

[0016] In one or more embodiments, the method for introducing the gene may be a method using a general gene recombination technique (for example, the method described in Michael R. Green & Joseph Sambrook, Molecular cloning, Cold Spring Harbor Laboratory Press). In one or more embodiments, examples of the gene introduction include gene introduction using a plasmid vector and integration into the chromosome of the microbial host. In the present disclosure, in one or more embodiments, "introducing a gene in an expressible manner" refers to introducing the gene in a manner that allows the introduced gene to be expressed in a transformant. In one or more embodiments, methods for introducing a gene in an expressible manner include a method of introducing the gene together with an expression regulatory sequence such as a promoter that can induce enhanced expression of the gene. In one or more embodiments, the gene introduced together with a promoter that can induce enhanced expression may be a single gene or an operon that can express multiple genes. In one or more embodiments, examples of promoters that can induce enhanced expression of genes include, but are not limited to, the T7 promoter.

[0017] In the present disclosure, the term "homologue" refers to a similar amino acid sequence or nucleotide sequence, preferably one having substantially the same biological function. In one or more embodiments, "similar amino acid sequence or nucleotide sequence" in the present disclosure refers to a sequence identity of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more.

[0018] In the present disclosure, "90% or more identity" with respect to an amino acid sequence or a nucleotide sequence refers to at least 90% identity, and in one or more embodiments, refers to 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity.

[0019] In the present disclosure, "identity of amino acid sequence or nucleotide sequence" can be performed using readily available sequence comparison computer programs. In one or more embodiments, computer programs include the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), the BLAST package (Ausubel et al. (1999) ibid-Ch. 18), and FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410).

[0020] In the present disclosure, "stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. In one or more embodiments, stringent conditions include conditions under which highly identical base sequences hybridize with each other, but less identical base sequences do not hybridize with each other. In one or more embodiments, high identity between base sequences means an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more. In one or more embodiments, stringent conditions may be those described in "Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol. 2, p. 11.45." Specifically, hybridization may occur at a temperature 5 to 10°C lower than the melting temperature (Tm) of a perfect hybrid.

[0021] In one or more embodiments, the microbial host may be not only a wild-type bacterium but also an artificially transformed bacterium. In one or more embodiments, the microbial host is not particularly limited and may include Escherichia coli (bacteria of the genus Escherichia, particularly Escherichia coli), coryneform bacteria, solvent-tolerant bacteria, yeast, or the like. In one or more embodiments, the microbial host may be Escherichia coli (bacteria of the genus Escherichia, particularly Escherichia coli), such as the E. coli BL21(DE3)ΔiscR strain. Coryneform bacteria are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), and are not particularly limited as long as they grow under normal aerobic conditions. In one or more embodiments, coryneform bacteria include bacteria of the genus Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus. In one or a plurality of embodiments, examples of solvent-resistant bacteria include Pseudomonas putida S12, Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas alcaligenes, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas oleovorans, Pseudomonas sp., Rhodococcus erythropolis, Rhodococcus opacus, Burkholderia cepacia, and Paenibacillus illinoisensis.

[0022] When the microbial host is Escherichia coli, in one or more embodiments, the genes encoding the enzyme subunits Hyd-1, Hyd-2, and Hyd-3 of the [NiFe]-hydrogenases (Hyd-1, Hyd-2, Hyd-3, and Hyd-4) in the host Escherichia coli may be disrupted or deleted. Genes encoding the enzyme subunits of [NiFe]-hydrogenase (Hyd-1, Hyd-2, Hyd-3, and Hyd-4) as well as factors regulating their enzyme expression, such as transcription and maturation, have been identified in Escherichia coli. Hyd-3 has been confirmed to play a central role in hydrogen production, while Hyd-1 and Hyd-2 have been confirmed to be primarily responsible for hydrogen uptake. In one or more embodiments, an example of a gene regulating the expression (or activity) of [FeFe]-hydrogenase is the iscR gene. Thus, when the microbial host is E. coli, in one or more embodiments, the iscR gene of E. coli may be disrupted or deleted.

[0023] [Transformants] In one aspect, the present disclosure relates to a transformant for producing hydrogen, which is obtained by introducing the following genes (A), (B), and (C) into the transformant in an expressible manner: (A) a gene encoding an electron-branching [FeFe]-hydrogenase or its homolog; (B) a gene encoding a [FeFe]-hydrogenase maturation factor or its homolog; (C) A gene encoding ferredoxin or its homologue.

[0024] As shown in Figure 1, the transformant of the present disclosure can produce hydrogen (H2) in vivo using sugars such as glucose as a raw material through gene (A) (EbhABC) and gene (B) (HydEFG), and can also improve hydrogen productivity through gene (C) (pfo). Furthermore, hydrogen can be produced more efficiently by introducing a gene capable of expressing ferredoxin together with and / or in place of gene (C).

[0025] [Gene (A): a gene encoding an electron-branching [FeFe]-hydrogenase or its homolog] Gene (A) in the present disclosure encodes an electron-bifurcating [FeFe]-hydrogenase or a homolog thereof. In one or more embodiments, an [FeFe]-hydrogenase is an enzyme that has a dinuclear iron complex at its active center and catalyzes a reaction that synthesizes or decomposes hydrogen. In an electron-bifurcating enzyme, two electrons can be transferred via different pathways. In one or more embodiments, an electron-bifurcating [FeFe]-hydrogenase or a homolog thereof includes a ferredoxin-NADH-dependent electron-bifurcating [FeFe]-hydrogenase. In one or more embodiments, an electron-bifurcating [FeFe]-hydrogenase may be formed from two or more types of subunits. In one or more embodiments, an electron-bifurcating [FeFe]-hydrogenase or a homolog thereof includes a trimeric electron-bifurcating [FeFe]-hydrogenase homolog and a trimeric ferredoxin-NADH-dependent electron-bifurcating [FeFe]-hydrogenase homolog. In the present disclosure, gene (A) is preferably a trimeric electron-forking [FeFe]-hydrogenase homologue or a trimeric ferredoxin-NADH-dependent electron-forking [FeFe]-hydrogenase homologue, since these genes can produce hydrogen more efficiently.

[0026] In one or more embodiments, gene (A) of the present disclosure may be a single gene or a combination of two or more genes. In one or more embodiments, gene (A) of the present disclosure may have three genes encoding homologs of subunits that constitute the [FeFe]-hydrogenase enzyme complex. The polypeptides encoded by these three genes may form a heterotrimer to form a trimeric electron-forking [FeFe]-hydrogenase having electron-forking [FeFe]-hydrogenase activity. In one or more embodiments, gene (A) of the present disclosure preferably has three genes encoding homologs of subunits that constitute the [FeFe]-hydrogenase enzyme complex, in order to produce hydrogen more efficiently.

[0027] In one or more embodiments, whether the enzyme encoded by gene (A) has [FeFe]-hydrogenase activity can be evaluated by carrying out a hydrogen production reaction using a transformant prepared by expressing the gene to be evaluated into [NiFe]-hydrogenase-deficient Escherichia coli. Confirmation of hydrogen production or an increase in the amount of hydrogen produced indicates that the enzyme has [FeFe]-hydrogenase activity. Hydrogen production can be measured as described in the Examples section of the present specification.

[0028] The origin of gene (A) is not particularly limited, and in one or more embodiments, the gene may be heterologous to the host. The origin of gene (A) is not particularly limited, and examples thereof include the genus Desulfobulbus. In one or more embodiments, examples of the genus Desulfobulbus include Desulfobulbus propionicus, Desulfobulbus rimicarensis, Desulfobulbus alkaliphilus, Desulfobulbus elongatus, Desulfobulbus oligotrophicus, Desulfobulbus oralis, and Desulfobulbus rhabdoformis.

[0029] In one or more embodiments, the gene encoding the trimeric electron-branched [FeFe]-hydrogenase derived from Desulfobulbus propionicus may be a combination of a gene consisting of the nucleotide sequence shown in SEQ ID NO: 4, a gene consisting of the nucleotide sequence shown in SEQ ID NO: 5, and a gene consisting of the nucleotide sequence shown in SEQ ID NO: 6. In one or more embodiments, the gene may be a combination of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0030] In one or more embodiments, the gene (A) may be a gene having a gene (a1), a gene (b1), and a gene (c1). (a1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; (c1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0031] In one or more embodiments, gene (A) may be a gene comprising gene (a2), gene (b2), and gene (c2). In this embodiment, the polypeptides encoded by gene (a2), gene (b2), and gene (c2) each constitute subunits of an electron-branching [FeFe]-hydrogenase, and these polypeptides preferably form a trimer, and the resulting trimer preferably has electron-branching [FeFe]-hydrogenase activity. (a2) a gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 1, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 1; (b2) a gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 2, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 2; (c2) A gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 3, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 3.

[0032] In one or more embodiments, the gene (a1) or (a2) may be at least one gene selected from the group consisting of the following (a11) to (a14): (a11) a gene having the nucleotide sequence shown in SEQ ID NO: 4 or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 4; (a12) a gene having a nucleotide sequence that has an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more to the nucleotide sequence shown in SEQ ID NO: 4, or a gene consisting of a nucleotide sequence that has an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more to the nucleotide sequence shown in SEQ ID NO: 4; (a13) A gene having a nucleotide sequence represented by SEQ ID NO: 4, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions per unit of 100 nucleotides; (a14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 4.

[0033] In one or more embodiments, the gene (b1) or (b2) may be at least one gene selected from the group consisting of the following (b11) to (b14): (b11) a gene having the nucleotide sequence shown in SEQ ID NO: 5 or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 5; (b12) a gene having a nucleotide sequence that has an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more to the nucleotide sequence shown in SEQ ID NO: 5, or a gene consisting of a nucleotide sequence that has an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more to the nucleotide sequence shown in SEQ ID NO: 5; (b13) a gene having a nucleotide sequence represented by SEQ ID NO: 5, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions are present per unit of 100 nucleotides; (b14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 5.

[0034] In one or more embodiments, the gene (c1) or (c2) may be at least one gene selected from the group consisting of the following (c11) to (c14): (c11) a gene having the nucleotide sequence shown in SEQ ID NO: 6 or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 6; (c12) a gene having a nucleotide sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the nucleotide sequence shown in SEQ ID NO: 6, or a gene consisting of a nucleotide sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the nucleotide sequence shown in SEQ ID NO: 6; (c13) a gene having a nucleotide sequence represented by SEQ ID NO: 6, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions per unit of 100 nucleotides; (c14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 6.

[0035] [Gene (B): [FeFe]-hydrogenase maturation factor or its homolog] In the present disclosure, gene (B) encodes a [FeFe]-hydrogenase maturation factor or a homolog thereof. In one or more embodiments, gene (B) is a gene involved in the maturation of gene (A) (electron-forking [FeFe]-hydrogenase or a homolog thereof). Maturation factors are auxiliary proteins involved in the process of incorporating a metal cluster ([FeFe] cluster) necessary for the catalytic function of electron-forking [FeFe]-hydrogenase into the enzyme, activating the enzyme. These maturation factors may also be called metallochaperones. Maturation of electron-forking [FeFe]-hydrogenase typically requires three factors: a radical SAM domain protein (HydE), an [FeFe]-hydrogenase H-cluster maturation GTPase (HydF), and an [FeFe]-hydrogenase H-cluster radical SAM maturase (HydG).

[0036] In one or more embodiments, gene (B) in the present disclosure preferably includes a gene encoding a radical SAM domain protein (HydE), a gene encoding an [FeFe]-hydrogenase H-cluster maturation GTPase (HydF), and a gene encoding an [FeFe]-hydrogenase H-cluster radical SAM maturase (HydG), in order to complete the maturation of electron-branching [FeFe]-hydrogenase.

[0037] In one or more embodiments, the origin of gene (B) is not particularly limited and may be heterologous to the host. In one or more embodiments, the origin of gene (B) is not particularly limited and may be the same species as gene (A) or heterologous. The origin of gene (B) is not particularly limited and may be, for example, the genus Desulfobulbus. Examples of the genus Desulfobulbus are the same as those of gene (A).

[0038] In one or more embodiments, examples of the gene encoding the [FeFe]-hydrogenase maturation factor derived from Desulfobulbus propionicus include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 10, a gene consisting of the nucleotide sequence shown in SEQ ID NO: 11, and a gene consisting of the nucleotide sequence shown in SEQ ID NO: 12. In one or more embodiments, examples of the gene include a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8, and a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0039] In one or more embodiments, the gene (B) may be a gene having a gene (d1), a gene (e1), and a gene (f1). (d1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 7, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7; (e1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 8, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8; (f1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 9, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0040] In one or more embodiments, gene (B) may be a gene comprising gene (d2), gene (e2), and gene (f2). In this embodiment, the polypeptides encoded by gene (d2), gene (e2), and gene (f2) may function as maturation factors for the polypeptide encoded by gene (A) and / or electron-branching [FeFe]-hydrogenase. (d2) a gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 7, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 7; (e2) a gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 8, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 8; (f2) A gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 9, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 9.

[0041] In one or more embodiments, the gene (d1) or (d2) may be at least one gene selected from the group consisting of the following (d11) to (d14): (d11) a gene having the nucleotide sequence shown in SEQ ID NO: 10 or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 10; (d12) a gene having a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity to the nucleotide sequence shown in SEQ ID NO: 10, or a gene consisting of a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity to the nucleotide sequence shown in SEQ ID NO: 10; (d13) a gene having a nucleotide sequence represented by SEQ ID NO: 10, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions per unit of 100 nucleotides; (d14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 10.

[0042] In one or more embodiments, the gene (e1) or (e2) may be at least one gene selected from the group consisting of any one of the following genes (e11) to (e14): (e11) a gene having the nucleotide sequence shown in SEQ ID NO: 11 or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 11; (e12) a gene having a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity to the nucleotide sequence shown in SEQ ID NO: 11, or a gene consisting of a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity to the nucleotide sequence shown in SEQ ID NO: 11; (e13) a gene having a nucleotide sequence represented by SEQ ID NO: 11, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions per unit of 100 nucleotides; (e14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 11.

[0043] In one or more embodiments, the gene (f1) or (f2) may be at least one gene selected from the group consisting of the following (f11) to (f14): (f11) a gene having the nucleotide sequence shown in SEQ ID NO: 12 or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 12; (f12) a gene having a nucleotide sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the nucleotide sequence shown in SEQ ID NO: 12, or a gene consisting of a nucleotide sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the nucleotide sequence shown in SEQ ID NO: 12; (f13) a gene having a nucleotide sequence represented by SEQ ID NO: 12, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions per unit of 100 nucleotides; (f14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 12.

[0044] [Gene (C): a gene encoding ferredoxin or its homolog] The gene (C) in the present disclosure encodes a ferredoxin or a homolog thereof. Ferredoxin (or a homolog thereof) can function as an electron carrier in hydrogen production by electron-branching [FeFe]-hydrogenase.

[0045] In one or more embodiments, the origin of gene (C) is not particularly limited and may be heterologous to the host. In one or more embodiments, the origin of gene (C) is not particularly limited and may be the same species as gene (A) and / or (B) or may be heterologous. The origin of gene (C) is not particularly limited and may include, for example, the genus Desulfobulbus and the genus Clostridium. Examples of the genus Desulfobulbus are the same as those of gene (A). In one or more embodiments, examples of the genus Clostridium include Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium butyricum, Clostridium cellulovorans, Clostridium kluyveri, Clostridium ljungdahlii, Clostridium pasteurianum, Clostridium saccharobutylicum, Clostridium thermophilus, and Clostridium tyrobutyricum.

[0046] In one or more embodiments, examples of the gene encoding ferredoxin derived from Clostridium acetobutylicum include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 20 and a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15. In one or more embodiments, examples of the gene encoding ferredoxin (ferredoxin homolog) derived from Desulfobulbus propionicus include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 21, 22, 23, or 24, and a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19.

[0047] In one or more embodiments, the gene (C) may be the following gene (g1) or (g2), etc. In this embodiment, the polypeptide encoded by the gene (g2) is a ferredoxin or a ferredoxin homolog. (g1) a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 15, 16, 17, 18, or 19, or a gene encoding a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 15, 16, 17, 18, or 19; (g2) A gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 15, 16, 17, 18, or 19, or a gene encoding a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 15, 16, 17, 18, or 19.

[0048] In one or more embodiments, the gene (g1) or (g2) may be at least one gene selected from the group consisting of the following (g11) to (g14): (g11) a gene having the nucleotide sequence shown in SEQ ID NO: 20, 21, 22, 23, or 24, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 20, 21, 22, 23, or 24; (g12) a gene having a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity to the nucleotide sequence shown in SEQ ID NO: 20, 21, 22, 23, or 24, or a gene consisting of a nucleotide sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity to the nucleotide sequence shown in SEQ ID NO: 20, 21, 22, 23, or 24; (g13) a gene having a nucleotide sequence represented by SEQ ID NO: 20, 21, 22, 23, or 24, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, and / or additions per unit of 100 nucleotides; (g14) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 20, 21, 22, 23 or 24.

[0049] [Gene (D): A gene encoding pyruvate ferredoxin oxidoreductase] In order to enable more efficient hydrogen production, in one or more embodiments, the transformant of the present disclosure may further be introduced in an expressible manner with a gene (D, Pfo gene) encoding pyruvate ferredoxin oxidoreductase (EC:1.2.7.1) instead of or together with gene (C). Pyruvate ferredoxin oxidoreductase is an enzyme that catalyzes the reaction of oxidizing pyruvate to produce acetyl-CoA.

[0050] In one or more embodiments, the origin of gene (D) is not particularly limited and may be the same species as or different from the host. The origin of gene (D) is not particularly limited and examples thereof include Escherichia coli (bacteria of the genus Escherichia, particularly Escherichia coli).

[0051] In one or more embodiments, examples of the gene encoding the pyruvate ferredoxin oxidoreductase derived from Escherichia coli (EcPfo gene) include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 14 and a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0052] In one or more embodiments, the gene (D) may be the following gene (h1) or (h2). In this embodiment, the polypeptide encoded by the gene (h2) may have the activity of oxidizing pyruvate to produce acetyl-CoA. (h1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 13, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13; or (h2) A polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 13, or a polypeptide consisting of an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identical to the amino acid sequence shown in SEQ ID NO: 13.

[0053] In one or more embodiments, the gene (h1) or (h2) may be at least one gene selected from the group consisting of the following (h11) to (h14): (h11) a gene having the nucleotide sequence shown in SEQ ID NO: 14, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 14; (h12) a gene having a nucleotide sequence that has an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more to the nucleotide sequence shown in SEQ ID NO: 14, or a gene consisting of a nucleotide sequence that has an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more to the nucleotide sequence shown in SEQ ID NO: 14; (h13) A gene having a base sequence represented by SEQ ID NO: 14, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base deletions, substitutions, and / or additions per unit, with 100 bases being one unit; (h14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 14.

[0054] In another aspect, the present disclosure relates to a transformant for producing hydrogen, which is obtained by expressibly introducing gene (a1), gene (b1), gene (c1), gene (d1), gene (e1), gene (f1), and (D) a gene encoding pyruvate ferredoxin oxidoreductase into a microbial host.

[0055] In another aspect, the present disclosure relates to a transformant for producing hydrogen, obtained by expressibly introducing gene (a2), gene (b2), gene (c2), gene (d2), gene (e2), gene (f2), and (D) a gene encoding pyruvate ferredoxin oxidoreductase into a microbial host, wherein the polypeptides encoded by gene (a2), gene (b2), and gene (c2) can form trimers and have electron-branched [FeFe]-hydrogenase activity, and the polypeptides encoded by gene (d2), gene (e2), and gene (f2) can have the function of maturing [FeFe]-hydrogenase.

[0056] In one or more embodiments, the transformant of the present disclosure may have a competing NADH utilization pathway disrupted in the host in order to enable more efficient hydrogen production. In one or more embodiments, examples of competing NADH utilization pathways include a lactic acid production pathway, a succinic acid production pathway, and an ethanol production pathway. In one or more embodiments, the transformant of the present disclosure may have at least one of the three pathways, the lactic acid production pathway, the succinic acid production pathway, and the ethanol production pathway, disrupted in order to enable more efficient hydrogen production, and all three pathways may be disrupted. In one or more embodiments, the gene in the lactic acid production pathway may be a gene encoding lactate dehydrogenase (e.g., the ldhA gene). In one or more embodiments, the gene in the succinic acid production pathway may be a gene encoding an enzyme having fumarate reductase activity (e.g., the frdB gene). In one or more embodiments, the gene in the ethanol production pathway may be a gene encoding acetaldehyde dehydrogenase / alcohol dehydrogenase activity (e.g., the adhE gene). In order to enable the transformant of the present disclosure to produce hydrogen more efficiently, in one or more embodiments, part or all of the gene encoding lactate dehydrogenase (e.g., the ldhA gene), the gene encoding acetaldehyde dehydrogenase / alcohol dehydrogenase activity (e.g., the adhE gene), and / or the gene encoding an enzyme having fumarate reductase activity (e.g., the frdB gene) in the host may be disrupted or deleted.

[0057] In order to enable the transformant of the present disclosure to produce hydrogen more efficiently, in one or more embodiments, part or all of the genes encoding transcription factors of the iron-sulfur cluster biosynthetic genes in the host may be disrupted or deleted.

[0058] In one aspect, the transformant according to the present disclosure may be modified to enhance each metabolic pathway by enhancing the expression of the various metabolic pathway genes described above or by highly activating enzyme functions through the use of heterologous (mutated) genes, for example.

[0059] [Vector / Plasmid] In another aspect, the present disclosure relates to a vector for introducing the above-mentioned gene (A) into a microorganism, a vector for introducing the above-mentioned gene (B) into a microorganism, a vector for introducing the above-mentioned gene (C) into a microorganism, and / or a vector for introducing the above-mentioned gene (D) into a microorganism. In another aspect, the present disclosure relates to a vector for introducing at least two or more genes selected from the group consisting of the above-mentioned gene (A), gene (B), gene (C), and gene (D). In one or more embodiments, the vector of the present disclosure can be used to produce the transformant of the present disclosure. The vector of the present disclosure is not particularly limited and may be, for example, a plasmid.

[0060] The above-described genes can be introduced into a microbial host by amplifying the above-described genes by PCR, cloning them into an appropriate vector that can be amplified in a microbial host such as E. coli, and transforming the microbial host with the vector. In one or more embodiments, examples of promoters include T7 promoter, lac promoter, tac promoter, and trc promoter.

[0061] [Creation of transformants] transformation The transformation method can be any known method without limitation, and in one or more embodiments, known methods include the calcium chloride / rubidium chloride method, the calcium phosphate method, DEAE-dextran-mediated transfection, and electroporation (electric pulse method).

[0062] Microbial growth In one or more embodiments, the transformant according to the present disclosure is preferably cultured and grown under aerobic conditions prior to reaction in a reaction solution. In one or more embodiments, the culture may be performed by liquid culture such as shaking culture, jar fermenter culture, or tank culture, or by solid culture. In one or more embodiments, the culture medium may be a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, and / or other nutrients. When the host is Escherichia coli, in one or more embodiments, the culture medium may be LB medium or the like. In one or more embodiments, the culture temperature is about 15°C to 40°C, or 30°C to 40°C. In one or more embodiments, the pH of the culture medium is 5 to 8 or 6 to 8. In one or more embodiments, the culture time is about 12 hours to 4 days, or 12 to 48 hours.

[0063] In one or more embodiments, examples of carbon sources include carbohydrates or sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; and alcohols such as ethanol and propanol. Hydrocarbons such as normal paraffin can also be used if desired. One carbon source may be used alone, or two or more carbon sources may be used in combination. In one or more embodiments, the concentration of the carbon source in the growth medium is approximately 0.1 (w / v%) to 10 (w / v%).

[0064] In one or more embodiments, examples of the nitrogen source include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, as well as urea, aqueous ammonia, sodium nitrate, and potassium nitrate. In one or more embodiments, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolysates, and amino acids can also be used. One nitrogen source may be used alone, or two or more may be used in combination. The concentration of the nitrogen source in the growth medium varies depending on the nitrogen compound used, but in one or more embodiments, it is approximately 0.1 (w / v%) to 10 (w / v%).

[0065] In one or more embodiments, examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, ammonium carbonate, and calcium carbonate. One type of inorganic salt may be used alone, or two or more types may be used in combination. The concentration of inorganic salts in the growth medium varies depending on the inorganic salt used, but in one or more embodiments, it is approximately 0.01 (w / v%) to 1 (w / v%).

[0066] In one or more embodiments, examples of other nutritional substances include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, hydrolyzed skim soybeans with hydrochloric acid, and extracts of animals, plants, or microbial cells, or decomposition products thereof. The concentration of the nutritional substances in the medium varies depending on the nutritional substances used, but in one or more embodiments, it is about 0.1 (w / v%) to 10 (w / v%). Vitamins may be added as needed. In one or more embodiments, examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0067] [Hydrogen production methods] In another aspect, the present disclosure relates to a method for producing hydrogen. In one aspect, the production method of the present disclosure includes a step of culturing the transformant of the present disclosure in a reaction solution containing sugars to produce hydrogen.

[0068] The reaction solution may contain sugars. In one or more embodiments, the reaction solution may be, but is not particularly limited to, water containing sugars, a buffer solution, an inorganic salt medium, or the above-mentioned medium. In one or more embodiments, the reaction solution may contain nutrients other than sugars, such as vitamins, yeast extract, and dry yeast.

[0069] In one or more embodiments, examples of sugars include glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol. In one or more embodiments, the concentration of sugars in the reaction solution is about 0.1 (w / v%) to 20 (w / v%), and preferably 1 (w / v%) to 20 (w / v%) or 5 (w / v%) to 20 (w / v%).

[0070] In one or more embodiments, examples of the buffer solution include phosphate buffer, Tris buffer, MOPS buffer, etc. In one or more embodiments, the concentration of the buffer solution is about 10 mM to 150 mM.

[0071] In one or more embodiments, the inorganic salts medium may include a medium containing one or more inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, calcium carbonate, urea, ammonium sulfate, and ferrous sulfate. Among these, a medium containing urea, ammonium sulfate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, and / or ferrous sulfate is preferred. The concentration of inorganic salts in the inorganic salts medium varies depending on the inorganic salt used, but in one or more embodiments, the concentration may be about 0.01 (w / v%) to 1 (w / v%).

[0072] Reaction conditions In one or more embodiments, the reaction conditions are preferably anaerobic conditions.

[0073] In one or more embodiments, the reaction temperature (the survival temperature of the transformant during the reaction) is about 15°C to 50°C. In one or more embodiments, the pH of the reaction solution is about 6 to 8. During the reaction, it is preferable to carry out the reaction while controlling the pH of the reaction solution to near neutral, particularly 6 to 7, using an aqueous ammonia solution, an aqueous sodium hydroxide solution, or the like with a pH controller (for example, Model DT-1023, manufactured by Able Co., Ltd.). In one or more embodiments, the reaction time is about 1 to 6 days, and preferably about 1 to 2 days. In one or more embodiments, the culture may be performed by any of batch, fed-batch, and continuous methods, with the batch method being preferred.

[0074] The present disclosure further relates to one or more of the following embodiments. [1] A transformant for producing hydrogen, obtained by expressibly introducing the following genes (A), (B), and (C) into a microbial host: (A) a gene encoding an electron-branching [FeFe]-hydrogenase or its homolog; (B) a gene encoding a [FeFe]-hydrogenase maturation factor or its homolog; (C) A gene encoding ferredoxin or its homologue. [2] The transformant according to [1], further comprising (D) a gene encoding pyruvate ferredoxin oxidoreductase introduced therein in an expressible manner. [3] The transformant according to [1] or [2], wherein the electron-branching [FeFe]-hydrogenase is a trimeric electron-branching [FeFe]-hydrogenase. [4] The transformant according to any one of [1] to [3], wherein the gene (C) encoding ferredoxin or a homolog thereof is a gene derived from the genus Clostridium or Desulfobulbus. [5] The transformant according to any one of [2] to [4], wherein the gene encoding the pyruvate ferredoxin oxidoreductase (D) is a gene derived from Escherichia coli. [6] A transformant for producing hydrogen, obtained by expressibly introducing the following gene (a1), the following gene (b1), the following gene (c1), the following gene (d1), the following gene (e1), the following gene (f1), and (D) a gene encoding pyruvate ferredoxin oxidoreductase into a microbial host: (a1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; (b1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (c1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3; (d1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 7; (e1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 8; (f1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 9. [7] A transformant for producing hydrogen, obtained by expressibly introducing the following genes into a microbial host: (a2), (b2), (c2), (d2), (e2), (f2), and (D) a gene encoding pyruvate ferredoxin oxidoreductase, The polypeptide encoded by gene (a2), the polypeptide encoded by gene (b2), and the polypeptide encoded by gene (c2) can form a trimer and have electron-branching [FeFe]-hydrogenase activity; and A transformant, wherein the polypeptide encoded by gene (d2), the polypeptide encoded by gene (e2), and the polypeptide encoded by gene (f2) have the function of maturing [FeFe]-hydrogenase. (a2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1; (b2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2; (c2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3; (d2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 7; (e2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 8; (f2) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:9. [8] The gene (a1) or (a2) is any one of the following genes (a11) to (a14), and / or The gene (b1) or (b2) is any one of the following genes (b11) to (b14): The gene (c1) or (c2) is any one of the following genes (c11) to (c14), and / or The gene (d1) or (d2) is any one of the following genes (d11) to (d14), and / or The gene (e1) or (e2) is any one of the following genes (e11) to (e14), and / or The transformant according to [6] or [7], wherein the gene (f1) or (f2) is any one of the genes (f11) to (f14) below: (a11) a gene having the nucleotide sequence shown in SEQ ID NO: 4; (a12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 4; (a13) a gene having a nucleotide sequence represented by SEQ ID NO: 4, with 1 to 10 base deletions, substitutions, and / or additions per unit of 100 bases; (a14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 4; (b11) a gene having the nucleotide sequence shown in SEQ ID NO: 5; (b12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 5; (b13) a gene having a nucleotide sequence represented by SEQ ID NO: 5, with 100 nucleotides per unit, and with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (b14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5; (c11) a gene having the nucleotide sequence shown in SEQ ID NO: 6; (c12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 6; (c13) a gene having a nucleotide sequence represented by SEQ ID NO: 6, with 100 nucleotides per unit, and with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (c14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6; (d11) a gene having the nucleotide sequence shown in SEQ ID NO: 10; (d12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 10; (d13) a gene having a nucleotide sequence represented by SEQ ID NO: 10, in which 100 nucleotides constitute one unit and in which 1 to 10 nucleotides have been deleted, substituted, and / or added per unit; (d14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10; (e11) a gene having the nucleotide sequence represented by SEQ ID NO: 11; (e12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 11; (e13) a gene having a nucleotide sequence represented by SEQ ID NO: 11, with 100 nucleotides per unit, and with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (e14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 11; (f11) a gene having the nucleotide sequence represented by SEQ ID NO: 12; (f12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 12; (f13) a gene having a nucleotide sequence represented by SEQ ID NO: 12, in which 100 nucleotides constitute one unit and in which 1 to 10 nucleotides have been deleted, substituted, and / or added per unit; (f14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 12. [9] The pyruvate ferredoxin oxidoreductase A polypeptide having the amino acid sequence set forth in SEQ ID NO: 13, or The transformant according to any one of [2] to [8], which is a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 13.

[10] The gene encoding the pyruvate ferredoxin oxidoreductase (D) is A gene having the base sequence shown in SEQ ID NO: 14, A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 14. A gene having a base sequence in which 1 to 10 bases are deleted, substituted, inserted and / or added per unit of 100 bases in the base sequence shown in SEQ ID NO: 14, or A transformant according to any one of [2] to [9], which is a gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 14.

[11] The transformant according to any one of [2] to

[10] , further comprising (C) a gene encoding ferredoxin or a homolog thereof introduced therein in an expressible manner.

[12] The ferredoxin or a homolog thereof A polypeptide having the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; or A polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. The transformant according to any one of [1] to

[11] ,

[13] The gene (C) encoding ferredoxin or a homolog thereof is A gene having a nucleotide sequence represented by SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or A gene having a base sequence in which 1 to 10 bases are deleted, substituted, inserted and / or added per unit of 100 bases in the base sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. A transformant according to any one of [1] to

[12] , which is a gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[14] The transformant according to any one of [1] to

[13] , in which at least one gene selected from the group consisting of a gene encoding lactate dehydrogenase, a gene encoding acetaldehyde dehydrogenase, a gene encoding alcohol dehydrogenase activity, and a gene encoding an enzyme having fumarate reductase activity is disrupted.

[15] The transformant according to any one of [1] to

[14] , in which a gene encoding a transcription factor of an iron-sulfur cluster biosynthetic gene is disrupted.

[16] A method for producing hydrogen, comprising the step of culturing the transformant according to any one of [1] to

[15] in a reaction solution containing sugars to produce hydrogen.

[0075] The present disclosure will be further described below using examples, but the present disclosure should not be construed as being limited to the following examples. [Example]

[0076] [Construction of strains (strains into which gene expression plasmids have been introduced)] (1) Preparation and acquisition of chromosomal DNA Chromosomal DNA of Desulfobulbus propionicus (DSMZ), Clostridium acetobutylicum (ATCC), and Escherichia coli BL21(DE3) (Novagen) was prepared using a DNA genome extraction kit (illustra bacteria genomicPrep Mini Spin Kit, Cytiva) after culturing the strains according to the information provided by the institution providing the strains.

[0077] (2) Construction of E. coli gene disruptant strains The target genes (iscR, frdB, ldhA, and adhE genes) were disrupted in the host strain Escherichia coli BL21(DE3). Disruption of the target genes in E. coli was performed using the Quick and Easy Escherichia coli Gene Deletion Kit (Gene Bridges), which modifies the E. coli chromosome by Red / ET recombination. PCR was used to create a DNA fragment for homologous recombination, in which homologous sequences of the disrupted gene were added to both ends of an FRT-PGK-gb2-neo-FRT cassette (Gene Bridges) containing a kanamycin resistance gene. The kanamycin resistance gene cassette, which had been inserted into the chromosome by Red / ET recombination, was then removed by FLP recombination. Disruption of the target gene was confirmed by PCR using the resulting gene-disrupted strain as a template. The following E. coli gene disruptant strains were constructed: an E. coli BL21(DE3) strain with the iscR gene disrupted (HC strain), an E. coli BL21(DE3) strain with the iscR and frdB genes disrupted, an E. coli BL21(DE3) strain with the iscR and ldhA genes disrupted, and an E. coli BL21(DE3) strain with the iscR and adhE genes disrupted. iscR gene The base sequences of the primers used to amplify a DNA fragment for disrupting the iscR gene, which encodes a transcription factor for iron-sulfur cluster biosynthesis genes, are as follows: 5'-TTTTACAATAAAAAACCCCGGGCAGGGGCGAGTTTGAGGTGAAGTAAGACAATTAACCCTCACTAAAGGGCG-3' (SEQ ID NO: 48) 5'-CCGTGTTTACGGAGTATTTAGCACTCCGGCCTGATTCTGAATTCTTTTTATAATACGACTCACTATAGGGCTC-3' (SEQ ID NO: 49) frdB gene The base sequences of the primers used to amplify a DNA fragment for disrupting the frdB gene encoding the fumarate reductase subunit involved in succinic acid production are as follows: 5'-AAGCAGCCAATAAGAAGGAGAAGGCGAATGGCTGAGATGAAAAACCTGAAAATTGAGAATTAACCCTCACTAAAGGGCGG-3' (SEQ ID NO: 50) 5′-GATTTGGATTAATCATCTCAGGCTCCTTACCAGTACAGGGCAACAAACAGGATTACTAATACGACTCACTATAGGGCTCG-3′ (SEQ ID NO: 51) ldhA gene The base sequences of the primers used to amplify a DNA fragment for disrupting the ldhA gene encoding lactate dehydrogenase involved in lactic acid production are as follows: 5'-TTTTGTAAAATATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTAATTAACCCTCACTAAAGGGCGG-3' (SEQ ID NO: 52) 5'-ATTGGGGATTATCTGAATCAGCTCCCCTGGGTTGCAGGGGAGCGGCAAGATAATACGACTCACTATAGGGCTC-3' (SEQ ID NO: 53) adhE gene The base sequences of the primers used to amplify the DNA fragment for disrupting the adhE gene encoding acetaldehyde dehydrogenase / alcohol dehydrogenase involved in ethanol production are as follows: 5′-AAAGTTTAACATTATCAGGAGAGCATTATGGCTGTTACTAATGTCGCTGAACTTAACGGCTGTGCAGGTCGTAAATC-3′ (SEQ ID NO: 54) 5'-TTTAGTTGCCAGACAGCGCTACTGATTAAGCGGATTTTTTCGCTTTTTTCTCAGCTAATACGACTCACTATAGGGCTCG-3' (SEQ ID NO: 55)

[0078] (3) Construction of gene expression plasmids - Construction of DpEbhABC expression plasmid (pCDFDpEbhABC) A DNA fragment containing the genes encoding the trimeric electron-branching hydrogenase homolog (DpEbhABC) from Desulfobulbus propionicus (Despr_1599 (SEQ ID NO: 6), Despr_1600 (SEQ ID NO: 5), and Despr_1601 (SEQ ID NO: 4)) was amplified by PCR and cloned into the NdeI / XhoI sites of the pCDFDuet®-1 vector (Merck) to generate a T7 promoter-driven DpEbhABC expression plasmid (pCDFDpEbhABC). The nucleotide sequences of the primers used to amplify the DpEbhABC gene are as follows: 5'-GGCCCCATATGACCACACTTGAGCAGGA-3' (SEQ ID NO: 26) 5'-GGCCCCTCGAGTCAGCAGGCGTCGGCCTG-3' (SEQ ID NO: 27) - Construction of SoHydGXFE expression plasmid (pETSoHydGXFE) A DNA fragment containing the gene encoding the [FeFe]-hydrogenase maturation factor (SoHydGXFE) (SEQ ID NO: 25) from Shewanella oneidensis was amplified by PCR and cloned into the pETDuet®-1 vector (Merk) to generate a T7 promoter-driven SoHydGXFE expression plasmid (pETSoHydGXFE). The codons for hydGXEF were modified for expression in Escherichia coli (Thermo Fisher) and the DNA fragment containing the HydGX gene amplified from the DNA fragment was inserted into the NdeI / XhoI site, while the DNA fragment containing the HydEF gene was inserted into the NcoI / BamHI site. The T7 promoter was located upstream of each DNA fragment. The base sequences of the primers used to amplify the SoHydGX gene are as follows: 5'-GGCCCCATATGAGCACCCATGAACATCA-3' (SEQ ID NO: 28) 5'-GGCCCCTCGAGTCATCGATTGAAGCCTTTAT-3' (SEQ ID NO: 29) The base sequences of the primers used to amplify the SoHydEF gene are as follows: SonHydE_Nco_Fw: 5′-GGCCCCCATGGGCATTACCCGTCCGAGTCC-3′ (SEQ ID NO: 30) SonHydF_Bam_Rv: 5′-GGCCCGGATCCTTACTGCTGCGGATTACGAT-3′ (SEQ ID NO: 31) - Construction of DpHydGFE expression plasmid (pETDpHydGFE) DNA fragments containing genes encoding [FeFe]-hydrogenase maturation factor homologs (DpHydG, DpHydF, and DpHydE) (Despr_1899 (SEQ ID NO: 12), Despr_1603 (SEQ ID NO: 11), and Despr_1604 (SEQ ID NO: 10)) from Desulfobulbus propionicus were amplified by PCR and cloned into the pETDuet®-1 vector (Merck) to generate a T7 promoter-driven DpHydGFE expression plasmid (pETDpHydGFE). The DNA fragment containing Despr_1899 (DpHydG gene) was inserted into the XbaI / BamHI site, and the DNA fragment containing Despr_1603-Despr_1604 (DpHydFE gene) was inserted into the NdeI / XhoI site, with the T7 promoter positioned upstream of each DNA fragment. The base sequences of the primers used to amplify the DpHydG gene are as follows: 5'-GGCCCTCTAGATAAGAAGGAGATATACCATGTTGATTGAAACTATGGA-3' (SEQ ID NO: 32) 5'-GGCCCGGATCCTTAGAAAAACAGATCGCGGC-3' (SEQ ID NO: 33) The base sequences of the primers used to amplify the DpHydFE gene are as follows: 5'-GGCCCCATATGTCGCAATCCGCACCCAA-3' (SEQ ID NO: 34) 5'-GGCCCCTCGAGCTATCCATCGCGTCCCTCCT-3' (SEQ ID NO: 35) - Construction of EcPfo expression plasmid (pACYCEcPfo) A DNA fragment containing the gene (SEQ ID NO: 14) encoding pyruvate ferredoxin oxidoreductase (EcPfo) was amplified from E. coli by PCR and cloned into the NdeI / XhoI sites of the pACYCDuet®-1 vector (Merck) to prepare a plasmid (pACYCEcPfo) for expression of EcPfo driven by the T7 promoter. The nucleotide sequences of the primers used to amplify the EcPfo gene are as follows: 5'-GGCCCCATATGATTACTATTGACGGTAATGG-3' (SEQ ID NO: 36) 5'-GGCCCCTCGAGTTAATCGGTGTTGCTTTTTTCC-3' (SEQ ID NO: 37) - Construction of EcPfo / CaFdx expression plasmid (pACYCEcPfo / CaFdx) A DNA fragment containing the gene encoding ferredoxin (CaFdx) (CA_C0303 (SEQ ID NO: 20)) from Clostridium acetobutylicum was amplified by PCR and cloned into the NcoI / BamHI site of the EcPfo expression plasmid (pACYCEcPfo) described above to generate the T7 promoter-driven EcPfo / CaFdx expression plasmid (pACYCEcPfo / CaFdx). The nucleotide sequences of the primers used to amplify CA_C0303 (CaFdx gene) are as follows: 5'-GGCCCCCATGGCATATAAAATAACAGACGCTTG-3' (SEQ ID NO: 38) 5'-GGCCCGGATCCTTACTCTTGAACTGGAGCTCC-3' (SEQ ID NO: 39) - Construction of EcPfo / Despr_0471 expression plasmid (pACYCEcPfo / Despr_0471) A DNA fragment containing the gene (Despr_0471 (SEQ ID NO: 21)) encoding a ferredoxin homolog from Desulfobulbus propionicus was amplified by PCR and cloned into the NcoI / BamHI site of the EcPfo expression plasmid (pACYCEcPfo) described above to prepare a T7 promoter-driven EcPfo / Despr_0471 expression plasmid (pACYCEcPfo / Despr_0471). The nucleotide sequences of the primers used to amplify the Despr_0471 gene are as follows: 5'-GGCCCCCATGGCACAGGAAGTACAGAT-3' (SEQ ID NO: 40) 5'-GGCCCGGATCCTCATTCTTTGCTGATACACC-3' (SEQ ID NO: 41) - Construction of EcPfo / Despr_1458 expression plasmid (pACYCEcPfo / Despr_1458) A DNA fragment containing the gene (Despr_1458 (SEQ ID NO: 22)) encoding a ferredoxin homolog from Desulfobulbus propionicus was amplified by PCR and cloned into the NcoI / BamHI site of the EcPfo expression plasmid (pACYCEcPfo) described above to prepare a T7 promoter-driven EcPfo / Despr_1458 expression plasmid (pACYCEcPfo / Despr_1458). The nucleotide sequences of the primers used to amplify the Despr_1458 gene are as follows: 5'-GGCCCCCATGGCGAAGCAGGTAAAAATCGA-3' (SEQ ID NO: 42) 5'-GGCCCGGATCCTTAGTCCTCCTCGTTGCTGA-3' (SEQ ID NO: 43) - Construction of EcPfo / Despr_2118 expression plasmid (pACYCEcPfo / Despr_2118) A DNA fragment containing the gene (Despr_2118 (SEQ ID NO: 23)) encoding a ferredoxin homolog from Desulfobulbus propionicus was amplified by PCR and cloned into the NcoI / BamHI site of the EcPfo expression plasmid (pACYCEcPfo) described above to prepare a T7 promoter-driven EcPfo / Despr_2118 expression plasmid (pACYCEcPfo / Despr_2118). The nucleotide sequences of the primers used to amplify the Despr_2118 gene are as follows: 5'-GGCCCCCATGGCGCCAAGGCCCCGCGCTCG-3' (SEQ ID NO: 44) 5'-GGCCCGGATCCTCACTCGTAGGAGATGCATC-3' (SEQ ID NO: 45) - Construction of EcPfo / Despr_2722 expression plasmid (pACYCEcPfo / Despr_2722) A DNA fragment containing the gene (Despr_2722 (SEQ ID NO: 24)) encoding a ferredoxin homolog from Desulfobulbus propionicus was amplified by PCR and cloned into the NcoI / BamHI site of the EcPfo expression plasmid (pACYCEcPfo) described above to prepare a T7 promoter-driven EcPfo / Despr_2722 expression plasmid (pACYCEcPfo / Despr_2722). The nucleotide sequences of the primers used to amplify the Despr_2722 gene are as follows: 5'-GGCCCCCATGGCGCCTGCGCTCGCCATCGA-3' (SEQ ID NO: 46) 5'-GGCCCGGATCCTCAGGCGTCGATATCCTCC-3' (SEQ ID NO: 47)

[0079] (4) Construction of a strain incorporating a plasmid for gene expression A strain (strain into which a gene expression plasmid has been introduced) was constructed using the above-mentioned E. coli gene-disrupted strain and gene expression plasmid. An overview of the introduced strain is shown in Table 1 below. [Table 1]

[0080] DpH0 strain (Reference Example 1) A strain (DpH0) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH0) was constructed by introducing the plasmid (pCDFDpEbhABC) for the expression of a trimeric electron-branching hydrogenase homologue from Desulfobulbus propionicus and the plasmid (pETSoHydGXFE) for the expression of a hydrogenase maturation factor from Shewanella oneidensis. DpH1 strain (Reference Example 2) A strain (DpH1) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH1) was constructed by introducing the plasmids (pCDFDpEbhABC) for the expression of a trimeric branched-chain hydrogenase homologue from Desulfobulbus propionicus and (pETDpHydGFE) for the expression of a hydrogenase maturation factor homologue from Desulfobulbus propionicus. DpH2 strain (Example 1) A strain (DpH2) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH2) was constructed by introducing the plasmids for the expression of a trimeric branched-chain hydrogenase homologue from Desulfobulbus propionicus (pCDFDpEbhABC), the expression of a hydrogenase maturation factor homologue from Desulfobulbus propionicus (pETDpHydGFE), and the expression of a pyruvate:ferredoxin oxidoreductase from E. coli (pACYCEcPfo). DpH3 strain (Example 2) A strain (DpH3) was constructed by using the iscR gene-disrupted strain (HC) of Escherichia coli BL21(DE3) as a host. The strain (DpH3) was transformed with the plasmids pCDFDpEbhABC for the expression of a trimeric electron-branching hydrogenase homologue from Desulfobulbus propionicus, pETDpHydGFE for the expression of a hydrogenase maturation factor homologue from Desulfobulbus propionicus, and pACYCEcPfo / CaFdx for the expression of E. coli pyruvate:ferredoxin oxidoreductase / Clostridium acetobutyricum ferredoxin. DpH4 strain (Example 3) A strain (DpH4) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH4) was constructed by introducing the following plasmids: a Desulfobulbus propionicus trimeric electron-branching hydrogenase homologue expression plasmid (pCDFDpEbhABC), a Desulfobulbus propionicus hydrogenase maturation factor homologue expression plasmid (pETDpHydGFE), and an E. coli pyruvate:ferredoxin oxidoreductase / Desulfobulbus propionicus ferredoxin homologue expression plasmid (pACYCEcPfo / Despr_0471). DpH5 strain (Example 4) A strain (DpH5) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH5) was constructed by introducing the following plasmids: a Desulfobulbus propionicus trimeric electron-branching hydrogenase homologue expression plasmid (pCDFDpEbhABC), a Desulfobulbus propionicus hydrogenase maturation factor homologue expression plasmid (pETDpHydGFE), and an E. coli pyruvate:ferredoxin oxidoreductase / Desulfobulbus propionicus ferredoxin homologue expression plasmid (pACYCEcPfo / Despr_1458). DpH6 strain (Example 5) A strain (DpH6) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH6) was constructed by introducing the following plasmids: a Desulfobulbus propionicus trimeric electron-branching hydrogenase homologue expression plasmid (pCDFDpEbhABC), a Desulfobulbus propionicus hydrogenase maturation factor homologue expression plasmid (pETDpHydGFE), and an E. coli pyruvate:ferredoxin oxidoreductase / Desulfobulbus propionicus ferredoxin homologue expression plasmid (pACYCEcPfo / Despr_2118). DpH7 strain (Example 6) A strain (DpH7) was constructed using the Escherichia coli BL21(DE3) strain (HC strain) in which the iscR gene was disrupted. The strain (DpH7) was constructed by introducing the following plasmids: a Desulfobulbus propionicus trimeric electron-branching hydrogenase homologue expression plasmid (pCDFDpEbhABC), a Desulfobulbus propionicus hydrogenase maturation factor homologue expression plasmid (pETDpHydGFE), and an E. coli pyruvate:ferredoxin oxidoreductase / Desulfobulbus propionicus ferredoxin homologue expression plasmid (pACYCEcPfo / Despr_2722). DpH8 strain (Example 7) A strain (DpH8) was constructed by using Escherichia coli BL21(DE3) with disruption of the iscR and frdB genes as a host. It was transformed with the plasmids pCDFDpEbhABC for the expression of a trimeric electron-branching hydrogenase homologue from Desulfobulbus propionicus, pETDpHydGFE for the expression of a hydrogenase maturation factor homologue from Desulfobulbus propionicus, and pACYCEcPfo / Despr_0471 for the expression of an E. coli pyruvate:ferredoxin oxidoreductase / Desulfobulbus propionicus ferredoxin homologue. DpH9 strain (Example 8) A strain (DpH9) was constructed by using Escherichia coli BL21(DE3) strain with disrupted iscR and ldhA genes as a host. The strain (DpH9) was constructed by introducing the plasmids for the expression of a trimeric electron-branching hydrogenase homologue from Desulfobulbus propionicus (pCDFDpEbhABC), the expression of a hydrogenase maturation factor homologue from Desulfobulbus propionicus (pETDpHydGFE), and the expression of a pyruvate:ferredoxin oxidoreductase / ferredoxin homologue from Desulfobulbus propionicus (pACYCEcPfo / Despr_0471). DpH10 strain (Example 9) A strain (DpH10) was constructed by using a strain of Escherichia coli BL21(DE3) in which the iscR and adhE genes were disrupted. The strain (DpH10) was constructed by introducing the plasmids for the expression of a trimeric electron-branching hydrogenase homologue from Desulfobulbus propionicus (pCDFDpEbhABC), the expression of a hydrogenase maturation factor homologue from Desulfobulbus propionicus (pETDpHydGFE), and the expression of a pyruvate:ferredoxin oxidoreductase / ferredoxin homologue from Desulfobulbus propionicus (pACYCEcPfo / Despr_0471).

[0081] [Enzyme activity measurement experiment (1) (reference experiment)] The hydrogenase activity of cell extracts from the HC strain (control) and the DpH0 strain (Reference Example 1) prepared as described above was compared. Enzyme activity analysis was performed as follows: The cells were inoculated into 20 mL of nutrient medium (per L: 5.0 g yeast extract, 10.0 g tryptone, 5.0 g glucose, 100 mM MOPS-NaOH (pH 7.4), 25 mM Na₂fumarate, 2 mM cysteine·HCl, 2 mM Fe·citrate, and 0.5 mM IPTG) in a test tube and placed in an anaerobic chamber (Coy Laboratory Products) containing 98.5% N₂ and 1.5% H₂ for 16 hours. Cells were harvested from the culture by centrifugation (7000 rpm, 3 min), suspended in 2 mL of 50 mM Tris-HCl (pH 7.5), and disrupted by sonication (SFX250; Branson; 25% power, 2 sec on / 10 sec off cycles for 5 min). The cell lysate was centrifuged (7000 rpm, 15 min), and the resulting supernatant was added to a hydrogenase reaction mixture (1 mL: 50 mM Tris-HCl (pH 7.5), 3 mM benzyl viologen, 100 μM sodium dithionite, 100 μL cell extract). The reduction of benzyl viologen was measured at 30 °C in a 1.5% H2 atmosphere using a spectrophotometer (GENESYS 10 S Bio; Thermo Fisher Scientific) by measuring the increase in absorbance at 578 nm. Benzyl viologen reduction activity was measured using a molar extinction coefficient (8600 M -1 cm -1 The above procedures were carried out in an anaerobic chamber. To calculate the specific enzyme activity, the protein concentration in the cell extract was measured using a protein assay dye reagent (Bio-Rad). Bovine serum albumin was used as the standard for protein quantification. The hydrogenase activity in the cell extract of the HC strain was below the detection limit, whereas the activity in the cell extract of the DpH0 strain was 0.33 μmol min -1 mg -1 Hydrogenase activity was detected. [Table 2]

[0082] [Enzyme activity measurement experiment (2) (reference experiment)] The hydrogenase activity of the cell extracts from the DpH0 strain (Reference Example 1) and the DpH1 strain (Reference Example 2) was measured in the same manner as above. Other results are shown below. [Table 3]

[0083] [Hydrogen production experiment (1)] The amount of hydrogen produced was evaluated by anaerobic culture using a sealed vial. The hydrogen production test culture method was as follows: 20 mL of nutrient medium (per L: 5.0 g yeast extract, 10.0 g tryptone, 5.0 g glucose, 100 mM MOPS-NaOH (pH 7.4), 2 mM Fe citrate) was placed in a 69 mL vial, and the DpH1 strain (Reference Example 2) and the DpH2 strain (Example 1) were inoculated and the OD 600 Static culture was performed at 37°C under aerobic conditions until the pH reached 0.5. IPTG (0.5 mM), cysteine·HCl (2 mM), and Na₂·fumarate (25 mM) were added, and the vial was sealed with a butyl rubber stopper and aluminum cap. The gas phase was then purged with nitrogen gas. The vial was then left to stand at 25°C for 16 to 24 hours, after which the hydrogen concentration in the vial gas phase was measured by gas chromatography. The results are shown in the table below. The hydrogen concentrations shown in the table are those after 16 hours of culture. More hydrogen was detected in the vial gas phase of the DpH1 strain than in the non-plasmid-introduced strain (HC strain), indicating that DpEbhABC generates hydrogen in vivo. The DpH2 strain (Example 1), into which E. coli pyruvate ferredoxin oxidoreductase (EcPfo) was introduced, had a hydrogen concentration in the vial gas phase that was more than twice as high as that of the DpH1 strain (Reference Example 2) without the enzyme. These results indicate that DpEbhABC accepts electrons from E. coli endogenous ferredoxin reduced by EcPfo to produce hydrogen. [Table 4]

[0084] [Hydrogen production experiment (2)] A hydrogen production experiment was conducted in the same manner as in Experiment (1) using strains (DpH3, DpH4, DpH5, DpH6, and DpH7) co-expressing Clostridium acetobutylicum ferredoxin (CaFdx) and four ferredoxin homologs (Despr_0471, Despr_1458, Despr_2118, and Despr_2722) found in the Desulfobulbus propionicus genome. The results are shown in the table below. The hydrogen concentrations shown in the table are those after 24 hours of culture. As shown in the table below, each strain exhibited improved hydrogen production compared to the DpH2 strain. In particular, the strain (DpH4) co-expressing Despr_0471 from Desulfobulbus propionicus exhibited a hydrogen concentration in the vial gas phase that was approximately 30% higher than that of the DpH2 strain. These results indicate that the CaFdx gene product, Despr_0471 gene product, Despr_1458 gene product, Despr_2118 gene product, or Despr_2722 gene product functions as a redox partner of EbhABC. [Table 5]

[0085] [Hydrogen Production Experiment (3)] A hydrogen production experiment was conducted in the same manner as in Experiment (1) using strains with a blocked succinate production pathway (DpH8), a blocked lactate production pathway (DpH9), and a blocked ethanol production pathway (DpH10), which compete with hydrogen production. The results are shown in the table below. The hydrogen concentrations shown in the table are those after 24 hours of culture. As shown in the table below, the hydrogen concentrations in the gas phase of the culture bottles of strains DpH8, DpH9, and DpH10 increased 24-fold, 36-fold, and 24-fold, respectively, compared to strain DpH4. Blocking the NADH oxidation pathway increased hydrogen production, suggesting that DpEbhABC can utilize NADH as an electron donor. These results indicate that DpEbhABC utilizes excess NADH generated by blocking the NADH utilization pathway for hydrogen production. [Table 6]

[0086] [Hydrogen Production Experiment (4)] We measured the hydrogen production per culture volume of strain DpH1, which was transformed with the trimeric electron-branching hydrogenase (DpEbhABC) from Desulfobulbus propionicus and the [FeFe]-hydrogenase maturation factor homolog (DpHydGFE), and strain DpH2, which was transformed with an expression plasmid for the pyruvate ferredoxin oxidoreductase (EcPfo) from Escherichia coli. Specifically, we performed a hydrogen production experiment similar to that described in Experiment (1). After 16 hours of culture, gas in a sealed vial was sampled with a syringe and the volume at 1 atmosphere was measured. The hydrogen concentration was measured by gas chromatography, and the amount of hydrogen was calculated based on 40.9 μmol of hydrogen per mL. In addition, using the DpH2 strain as a base, we co-expressed the Despr_0471 gene, which encodes a ferredoxin homologue from Desulfobulbus propionicus, and further performed hydrogen production experiments using the DpH8 strain, in which the succinate production pathway was blocked, the DpH9 strain, in which the lactate production pathway was blocked, and the DpH10 strain, in which the ethanol production pathway was blocked.We then measured the amount of hydrogen produced per culture volume from the gas contained in the sealed vials after 24 hours of culture, as described above. These results are shown in the table below. As shown in the table below, all strains showed hydrogen production that was approximately 2400% higher than that of the DpH2 strain, significantly exceeding that of the DpH2 strain. In particular, the DpH9 strain produced approximately 30% more hydrogen than the DpH8 and DpH10 strains, indicating that blocking the lactic acid production pathway is the most effective way to improve hydrogen production. [Table 7]

[0087] The names of the enzymes encoded by the genes shown in Figure 1 (a metabolic pathway diagram for explaining the hydrogen biosynthetic pathway) and their EC numbers are shown below. The "enzyme names" shown below are merely examples, and it goes without saying that there may be synonymous alternative names. The "enzymes" encoded by the "genes" shown below are merely examples, and it goes without saying that there may be "genes" that encode "enzymes" with multiple functions. [Table 8]

[0088] Below are the abbreviated compound names and their CAS numbers shown in Figure 1 (a metabolic pathway diagram for explaining the hydrogen biosynthesis pathway). It goes without saying that the "compound names" shown below are only examples, and that synonymous aliases may exist. It goes without saying that the "CAS numbers" shown below are not necessarily a comprehensive list. [Table 9]

Claims

1. A transformant for producing hydrogen, obtained by expressibly introducing the following genes (A), (B), and (C) into a microbial host: (A) a gene encoding an electron-branching [FeFe]-hydrogenase or a homolog thereof; (B) a gene encoding a [FeFe]-hydrogenase maturation factor or a homolog thereof; (C) A gene encoding ferredoxin or a homolog thereof.

2. The transformant according to claim 1, further comprising (D) a gene encoding pyruvate ferredoxin oxidoreductase introduced therein in an expressible manner.

3. The transformant according to claim 1, wherein the electron-branching [FeFe]-hydrogenase is a trimeric electron-branching [FeFe]-hydrogenase.

4. The transformant according to claim 1 , wherein the gene (C) encoding ferredoxin or a homolog thereof is a gene derived from the genus Clostridium or Desulfobulbus .

5. 3. The transformant according to claim 2, wherein the gene (D) encoding pyruvate ferredoxin oxidoreductase is a gene derived from Escherichia coli.

6. A transformant for producing hydrogen, obtained by expressibly introducing the following genes into a microbial host: gene (a1), gene (b1), gene (c1), gene (d1), gene (e1), gene (f1), and (D) a gene encoding pyruvate ferredoxin oxidoreductase: (a1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; (b1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (c1) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3; (d1) a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 7; (e1) a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 8; (f1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:

9.

7. A transformant for producing hydrogen, obtained by expressibly introducing the following genes into a microbial host: a gene (a2), a gene (b2), a gene (c2), a gene (d2), a gene (e2), a gene (f2), and (D) a gene encoding pyruvate ferredoxin oxidoreductase, The polypeptide encoded by gene (a2), the polypeptide encoded by gene (b2), and the polypeptide encoded by gene (c2) can form a trimer and have electron-branching [FeFe]-hydrogenase activity; and A transformant, wherein the polypeptide encoded by gene (d2), the polypeptide encoded by gene (e2), and the polypeptide encoded by gene (f2) have the function of maturing [FeFe]-hydrogenase. (a2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1; (b2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2; (c2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3; (d2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 7; (e2) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 8; (f2) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:

9.

8. The gene (a1) or (a2) is any one of the following genes (a11) to (a14): The gene (b1) or (b2) is any one of the following genes (b11) to (b14): The gene (c1) or (c2) is any one of the following genes (c11) to (c14), and / or The gene (d1) or (d2) is any one of the following genes (d11) to (d14): The gene (e1) or (e2) is any one of the following genes (e11) to (e14): The transformant according to claim 6 or 7, wherein the gene (f1) or (f2) is any one of the following genes (f11) to (f14): (a11) a gene having the base sequence represented by SEQ ID NO: 4; (a12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 4; (a13) a gene having a nucleotide sequence represented by SEQ ID NO: 4, with 1 to 10 base deletions, substitutions and / or additions per unit of 100 bases; (a14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 4; (b11) a gene having the nucleotide sequence shown in SEQ ID NO: 5; (b12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 5; (b13) a gene having a base sequence represented by SEQ ID NO: 5, with 100 bases as one unit, and with 1 to 10 base deletions, substitutions and / or additions per unit; (b14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5; (c11) a gene having the nucleotide sequence represented by SEQ ID NO: 6; (c12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 6; (c13) a gene having a nucleotide sequence represented by SEQ ID NO: 6, with 100 nucleotides per unit, and with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (c14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 6; (d11) a gene having the base sequence represented by SEQ ID NO: 10; (d12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 10; (d13) a gene having a nucleotide sequence represented by SEQ ID NO: 10, with 100 nucleotides per unit, and with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (d14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10; (e11) a gene having the base sequence represented by SEQ ID NO: 11; (e12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 11; (e13) a gene having a base sequence represented by SEQ ID NO: 11, with 100 bases as one unit, and with 1 to 10 base deletions, substitutions and / or additions per unit; (e14) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 11; (f11) a gene having the nucleotide sequence represented by SEQ ID NO: 12; (f12) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 12; (f13) a gene having a nucleotide sequence represented by SEQ ID NO: 12, with 100 nucleotides per unit, and with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (f14) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO:

12.

9. The pyruvate ferredoxin oxidoreductase A polypeptide having the amino acid sequence set forth in SEQ ID NO: 13, or 8. The transformant according to any one of claims 2, 5, 6 and 7, which is a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:

13.

10. The gene (D) encoding pyruvate ferredoxin oxidoreductase is A gene having the base sequence shown in SEQ ID NO: 14, A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 14, A gene having a base sequence in which 1 to 10 bases per unit of 100 bases are deleted, substituted, inserted and / or added in the base sequence shown in SEQ ID NO: 14, or 8. The transformant according to any one of claims 2, 5, 6 and 7, which is a gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO:

14.

11. The transformant according to claim 6 or 7, further comprising (C) a gene encoding ferredoxin or a homolog thereof introduced therein in an expressible manner.

12. The ferredoxin or a homolog thereof A polypeptide having the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19; or A polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO:

19.

6. The transformant according to claim 1, wherein

13. The gene (C) encoding ferredoxin or a homolog thereof is A gene having a base sequence represented by SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or A gene having a base sequence in which 1 to 10 bases are deleted, substituted, inserted and / or added per unit of 100 bases in the base sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:

24. A transformant described in any one of claims 1 to 5, which is a gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the base sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:

24.

14. 8. A transformant according to any one of claims 1 to 7, in which at least one gene selected from the group consisting of a gene encoding lactate dehydrogenase, a gene encoding acetaldehyde dehydrogenase, a gene encoding alcohol dehydrogenase activity, and a gene encoding an enzyme having fumarate reductase activity is disrupted.

15. The transformant according to any one of claims 1 to 7, wherein a gene encoding a transcription factor of the iron-sulfur cluster biosynthetic gene is disrupted.

16. A method for producing hydrogen, comprising a step of culturing the transformant according to any one of claims 1 to 7 in a reaction solution containing sugars to produce hydrogen.

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