Biotin-auxotrophic hydrogen-oxidizing bacterium mutant

Abstract

Provided is a hydrogen-oxidizing bacterium mutant in which at least one of the below-mentioned genes (1) to (4) is knockout, wherein the genes are located on the chromosome of a hydrogen-oxidizing bacterium. The hydrogen-oxidizing bacterium mutant is improved in terms of the efficiency of the production of a target substance. (1) An 8-amino-7-oxononanate synthase gene; (2) an 8-amino-7-oxononanate aminotransferase gene; (3) a desthiobiotin synthase gene; and (4) a biotin synthase gene. The hydrogen-oxidizing bacterium mutant may further have a foreign gene for producing a target substance. The target substance can be produced with high efficiency by culturing the hydrogen-oxidizing bacterium mutant using a biotin-containing culture medium to proliferate the hydrogen-oxidizing bacterium mutant and subsequently further culturing the hydrogen-oxidizing bacterium mutant using a biotin-depleted culture medium.

Description

Biotin-requiring hydrogen bacteria mutants

[0001] The present invention relates to a mutant of hydrogen bacteria that has been made biotin-requiring by disrupting a biotin biosynthesis gene, and to a method for producing compounds, proteins, etc. using this mutant.

[0002] The Paris Agreement, adopted in 2015, calls for rapid reductions in global greenhouse gas emissions. As part of its efforts to comply with this agreement, the Japanese government announced in April 2021 its goal of reducing greenhouse gas emissions, including carbon dioxide and methane, by 46% by 2030 compared to 2013 levels.

[0003] Globally, the majority of chemical production relies on petroleum as a raw material, resulting in problems such as increasing greenhouse gas emissions. Therefore, there is a need to move away from petroleum-based chemical production, and research and development into biorefineries that produce green chemicals from biomass is being actively conducted in various countries. However, converting biomass into sugars to obtain raw materials for microbial fermentation requires complex processes, posing a high cost issue. Furthermore, using biomass, which can be used as food or feed, in chemical production hinders the stable supply of food and feed. Furthermore, there is also the problem that consuming large amounts of biomass in chemical production actually leads to environmental destruction.

[0004] As part of research into decarbonization, gases such as carbon dioxide, methane, and carbon monoxide are attracting attention as carbon feedstocks with a higher degree of sustainability, and there is growing interest in technologies for producing valuable materials using microorganisms that utilize these gases. Among these, there are high hopes for fixing and effectively utilizing carbon dioxide, which contributes significantly to global warming. Fixing carbon dioxide to produce materials does not have the disadvantages of using sugars derived from biomass as a carbon feedstock.

[0005] Hydrogen-oxidizing bacteria (also known as "hydrogen bacteria") can grow using carbon dioxide as the sole carbon source, utilizing the chemical energy generated by the reaction of hydrogen and oxygen. They can produce chemical products using a mixture of oxygen, hydrogen, and carbon dioxide as raw materials, so they can efficiently organicize carbon dioxide and can be cultured in a simple medium.

[0006] For this reason, the present inventor has developed bioprocesses using hydrogen bacteria to produce various chemical substances and has obtained patents (e.g., Patent Document 1). However, further advances in bioprocesses using hydrogen bacteria require innovative improvements in substance production efficiency.

[0007] Patent No. 6450912

[0008] Chemistry and Biology 3 (11): 569-574.

[0009] An object of the present invention is to provide a mutant of hydrogen bacteria with improved production efficiency of a target substance, and a method for efficiently producing a target substance using the mutant.

[0010] The present inventors conducted extensive research to solve the above-mentioned problems and obtained the following findings. (i) Through genome analysis of the biotin biosynthetic metabolic pathway of a representative hydrogen bacterium, the genus Hydrogenophilus, the present inventors identified four genes, bioF, bioA, bioD, and bioB, as essential for biotin biosynthesis. They then constructed a mutant strain in which the bioB gene was disrupted by homologous recombination. (ii) This bioB-disrupted mutant strain exhibited biotin auxotrophy, requiring external biotin for growth. Growth was nearly abolished when biotin was depleted during cultivation. It has long been known that halting microbial growth by external factors induces phenomena known as autolysis and bacteriolysis, making it impossible to maintain substance production (Non-Patent Document 1). However, surprisingly, no autolysis or bacteriolysis of the bacterial cells was observed in this bioB-disrupted mutant. Furthermore, it was confirmed that key intracellular metabolic functions, including carbon dioxide uptake, were maintained. (iii) The bioF, bioA, and bioD genes involved in biotin biosynthesis, together with the bioB gene, control a series of metabolic reactions. Therefore, in the bioF, bioA, and bioD gene disruption mutants, as in the bioB disruption mutant, autolysis and lysis of the bacterial cells are not induced even when biotin is depleted, and major intracellular metabolic functions, including carbon dioxide uptake, are maintained. Therefore, it is reasonably predicted that efficient substance production can be achieved using carbon dioxide as the sole carbon source.

[0011] The present invention was completed based on the above findings and provides the following [1] to [3]. [1] A hydrogen bacterium mutant in which any one or more of the following genes (1) to (4) present on the chromosome of the hydrogen bacterium have been disrupted: (1) an 8-amino-7-oxononanoic acid synthase gene; (2) an 8-amino-7-oxononanoic acid aminotransferase gene; (3) a desthiobiotin synthase gene; and (4) a biotin synthase gene. [2] The hydrogen bacterium mutant according to [1], which has an exogenous gene for producing a target substance. [3] A method for producing a target substance, which comprises culturing the hydrogen bacterium mutant according to [1] or [2] in a biotin-deficient medium.

[0012] Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, solar, wind, geothermal, and other energy sources are being used in place of fossil fuels. However, in reality, even these energy sources have not been able to sufficiently curb the increase in carbon dioxide. Therefore, it is necessary to promote the fixation or resource recovery of emitted carbon dioxide. Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can be used to produce organic matter that can be used as food, feed, fuel, and other raw materials for chemical products. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve both the two problems of global warming caused by increased carbon dioxide and the difficulty in securing food, feed, fuel, and raw materials for chemical products.

[0013] Hydrogen bacteria, which can grow using carbon dioxide as their sole carbon source, can utilize the chemical energy generated by the reaction of hydrogen and oxygen. Because they can grow on a mixture of oxygen, hydrogen, and carbon dioxide, they can efficiently convert carbon dioxide into organic matter. Furthermore, they can be cultured in simple media that do not contain organic matter such as sugars. This has led to demand for the use of hydrogen bacteria to extract valuable resources from carbon dioxide. While hydrogen bacteria generally grow slowly, Hydrogenophilus bacteria grow at a significantly faster rate. The Mitsubishi Research Institute Bulletin No. 34, 1999, evaluated Hydrogenophilus bacteria, stating, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation capacity of plants, clearly demonstrating the high carbon dioxide fixation capacity of microorganisms." Because Hydrogenophilus bacteria possess the carbon dioxide fixation or intracellular metabolic capabilities that enable such high growth rates, they can produce substances with high efficiency even when their growth is suppressed.

[0014] Biotin is a substance necessary for maintaining the living body as a coenzyme for biotin enzymes involved in fatty acid metabolism such as carbon fixation, carbon transfer, and decarboxylation, sugar metabolism, branched-chain amino acid metabolism, etc. In particular, in microorganisms, biotin is involved in the synthesis of cell membranes, so if biotin biosynthesis is inhibited by an external factor, autolysis or bacteriolysis generally occurs unless biotin is supplied.

[0015] The biosynthetic pathway of biotin from pimelic acid in microorganisms is shown in Figure 1. 8-Amino-7-oxononanoic acid synthase (BioF) converts pimeloyl-CoA / ACP to 8-amino-7-oxononanoic acid, 8-amino-7-oxononanoic acid aminotransferase (BioA) converts 8-amino-7-oxononanoic acid to 7,8-diaminononanoic acid, desthiobiotin synthase (BioD) converts 7,8-diaminononanoic acid to desthiobiotin, and biotin synthase (BioB) converts desthiobiotin to biotin.

[0016] Hydrogen bacteria naturally possess enzyme genes that catalyze the biotin biosynthesis reaction from pimeloyl-CoA / ACP and do not require external biotin supply for growth. However, disruption of one or more of these enzyme genes can confer biotin auxotrophy. Although the hydrogen bacteria mutants of the present invention have one or more of the enzyme genes involved in biotin biosynthesis disrupted (particularly deleted), resulting in suppression of biotin biosynthesis, they do not undergo autolysis or bacteriolysis even when biotin supply is stopped, and major intracellular metabolic functions, including those related to carbon dioxide uptake, are maintained. Therefore, by creating transformants of the hydrogen bacteria mutants of the present invention by introducing metabolic enzyme genes or recombinant protein genes for target substance production and culturing the bacteria in a medium containing little or no biotin, the carbon dioxide taken up is not wasted for bacterial growth, and metabolic reactions to produce target substances and recombinant proteins can be achieved with high efficiency. Furthermore, by culturing the hydrogen bacteria mutant of the present invention in a medium containing little or no biotin, the absorbed carbon dioxide is not wasted in the growth of the bacterial cells, and target substances such as compounds and proteins can be produced with high efficiency through the expression of genes present in the genome of the hydrogen bacteria mutant.

[0017] FIG. 1 shows the biotin biosynthesis pathway of microorganisms.

[0018] The present invention is described in detail below. (1) Hydrogen bacterium mutant conferred with biotin auxotrophy The hydrogen bacterium mutant of the present invention is a mutant in which one or more of the following genes (1) to (4) that constitute the biotin biosynthetic system present on the chromosome of the host hydrogen bacterium are disrupted: (1) A gene (bioF) encoding a polypeptide with 8-amino-7-oxononanoic acid synthase activity that produces 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA. (2) A gene (bioA) encoding a polypeptide with 8-amino-7-oxononanoic acid aminotransferase activity that produces 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid. (3) A gene (bioD) encoding a polypeptide with desthiobiotin synthase activity that produces desthiobiotin from 7,8-diaminononanoic acid. (4) A gene (bioB) encoding a polypeptide with biotin synthase activity that produces biotin from desthiobiotin. These mutants are unable to biotin biosynthesize themselves or have reduced biotin biosynthesis, and therefore exhibit biotin auxotrophy, requiring external biotin supply for growth.

[0019] Disruption of Enzyme Genes Constituting the Biotin Biosynthesis System In the present invention, "disruption of the bioF gene, bioA gene, bioD gene, or bioB gene" refers to the reduction of the activity of the product of each gene to 50% or less of that of the parent strain before disruption. Preferably, the reduction is 10% or less, and particularly 1% or less. This results in a lower growth rate in a biotin-free medium compared to the parent strain. The activity of the gene product is evaluated by measuring the catalytic activity of the enzyme. A decrease in gene expression level reduces the activity of the gene product. Gene expression level can be measured by quantitative PCR (qPCR) or next-generation sequencing (NGS). Although a gene may be expressed normally even if part of its coding region or expression regulatory region is mutated, this also constitutes "gene disruption" as long as the activity of the gene product is 50% or less of that of the parent strain. Gene disruption can be achieved by mutating (by base deletion, substitution, insertion, addition, or a combination thereof) the coding region or gene expression regulatory region, such as the promoter region, of the gene on the chromosome of the hydrogen bacterium. Methods for gene disruption are well known, including gene knockout methods using homologous recombination, and methods involving the introduction of random gene mutations using mutagens and screening based on phenotypes. Although gene knockout using homologous recombination has not been known for hydrogen bacteria, particularly bacteria of the genus Hydrogenophilus, gene disruption in the present invention can be carried out by the procedure described in the Examples section.

[0020] The mutant of the present invention preferably has a mutation in one or more of the coding regions bioF, bioA, bioD, and bioB. Mutation of each coding region typically involves deleting the entire or partial region of each gene. Alternatively, a gene can be disrupted by inserting a nucleotide, oligonucleotide, or polynucleotide into the gene. Alternatively, the entire or partial region of each gene can be replaced with another nucleotide, oligonucleotide, or polynucleotide. When multiple nucleotide deletions, substitutions, and / or insertions are introduced, they may be introduced at a single site in the gene or at multiple dispersed sites.

[0021] The number of nucleotides to be deleted, substituted, or inserted is preferably 3 or more. It is also preferable to delete, replace, or insert 1% or more, more preferably 5% or more, more preferably 10% or more, and even more preferably 50% or more of the nucleotides of the entire gene. This ensures that each gene is disrupted. It is also possible to delete or replace the entire length of each gene (i.e., 100% of the nucleotides constituting each gene). The number of nucleotides to be inserted can be 100,000 or less.

[0022] The bioF of hydrogen bacteria is any one of the following genes (a1) to (a5): (a1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 1; (a2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 1, and encoding a polypeptide having 8-amino-7-oxononanoic acid synthase activity that produces 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA; (a3) ​​DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 2; (a4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 2, and having 8-amino-7-oxononanoic acid synthase activity that produces 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA; (a5) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 40, preferably 1 to 20, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, or preferably 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, and having 8-amino-7-oxononanoic acid synthase activity that produces 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA. SEQ ID NO: 1 is the nucleotide sequence of HPTL_1306, which is the bioF gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 2 is the amino acid sequence of the polypeptide encoded by HPTL_1306 of the Hydrogenophilus thermorteolus wild-type strain.

[0023] The bioA gene of the hydrogen bacteria is any one of the following genes (b1) to (b5). (b1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 3; (b2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity with the nucleotide sequence of SEQ ID NO: 3, and encoding a polypeptide having 8-amino-7-oxononanoic acid aminotransferase activity to produce 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid; (b3) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 4; (b4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity with SEQ ID NO: 4, and having 8-amino-7-oxononanoic acid aminotransferase activity to produce 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid; (b5) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 40, preferably 1 to 20, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, or preferably 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4, and having 8-amino-7-oxononanoic acid aminotransferase activity that produces 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid. SEQ ID NO: 3 is the base sequence of HPTL_1305, which is the bioA gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 4 is the amino acid sequence of the polypeptide encoded by HPTL_1305 of a Hydrogenophilus thermorteolus wild-type strain.

[0024] BioD of hydrogen bacteria is any one of the following genes (c1) to (c5): (c1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 5; (c2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 5, and encoding a polypeptide having desthiobiotin synthase activity to produce desthiobiotin from 7,8-diaminononanoic acid; (c3) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 6; (c4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 6, and having desthiobiotin synthase activity to produce desthiobiotin from 7,8-diaminononanoic acid; (c5) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 20, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, or preferably 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6, and which has desthiobiotin synthase activity that produces desthiobiotin from 7,8-diaminononanoic acid. SEQ ID NO: 5 is the nucleotide sequence of HPTL_1309, which is the bioD gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 6 is the amino acid sequence of the polypeptide encoded by HPTL_1309 of a Hydrogenophilus thermorteolus wild-type strain.

[0025] BioB of hydrogen bacteria is any one of the following genes (d1) to (d5): (d1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 7; (d2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 7, and encoding a polypeptide having biotin synthase activity that synthesizes biotin from desthiobiotin; (d3) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 8; (d4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 8, and having biotin synthase activity that synthesizes biotin from desthiobiotin; (d5) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 30, preferably 1 to 20, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, or preferably 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 8, and which has biotin synthase activity that synthesizes biotin from desthiobiotin. SEQ ID NO: 7 is the base sequence of HPTL_1304, which is the bioB gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 8 is the amino acid sequence of the polypeptide encoded by HPTL_1304 of a Hydrogenophilus thermorteolus wild-type strain.

[0026] In the present invention, the identity of the base sequence and amino acid sequence is a value calculated using GENETYX ver. 17 (manufactured by GENETYX Corporation).

[0027] The test polypeptide's 8-amino-7-oxononanoic acid synthase activity, which produces 8-amino-7-oxononanoic acid from pimeloyl-ACP / CoA, is confirmed by adding pimeloyl-CoA as a substrate to a cell lysate containing the test polypeptide, allowing the reaction to proceed, and quantifying the by-product CoA based on absorbance at 260 nm. Specific reaction conditions can be appropriately determined by those skilled in the art by referring to Applied and Environmental Microbiology, 84:e02084-17 (2018). Whether a test polypeptide has 8-amino-7-oxononanoate aminotransferase activity to produce 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid can be confirmed by adding 8-amino-7-oxononanoic acid as a substrate to a cell lysate containing the test polypeptide, allowing the reaction to proceed, derivatizing the 7,8-diaminononanoic acid in the reaction solution with orthophthalaldehyde, and quantifying the fluorescence intensity of the resulting derivative at an excitation wavelength of 410 nm and a measurement wavelength of 470 nm. Specific reaction conditions can be appropriately determined by those skilled in the art by referring to Analytical Biochemistry, 432(2):90-96 (2012). Whether a test polypeptide has desthiobiotin synthase activity to synthesize desthiobiotin from 7,8-diaminononanoic acid can be confirmed by adding 7,8-diaminononanoic acid as a substrate to a cell lysate containing the test polypeptide, allowing the reaction to proceed, separating the reaction solution by liquid chromatography, and quantifying the produced desthiobiotin based on absorbance at 260 nm. Specific reaction conditions can be appropriately determined by those skilled in the art with reference to Biochemistry, 49(31):6746-6760 (2010). Whether a test polypeptide has biotin synthase activity to synthesize biotin from desthiobiotin can be confirmed by adding desthiobiotin as a substrate to a cell lysate containing the test polypeptide, allowing the reaction to proceed, separating the reaction solution by liquid chromatography, and quantifying the produced biotin based on absorbance at 260 nm. Specific reaction conditions can be appropriately determined by those skilled in the art with reference to Biochemistry, 49(46):9985-9996 (2010).

[0028] Examples of preferred hydrogen bacteria that can be used in the present invention include Pseudonocardia bacteria such as Pseudonocardia autotrophica; Hydrogenobacter bacteria such as Hydrogenobacter hydrogenophilus; Hydrogenophilus bacteria such as Hydrogenophilus thermoluteolus and Hydrogenophilus thiooxidans; Paracoccus bacteria such as Paracoccus denitrificans and Paracoccus pantotrophus; Xanthobacter bacteria such as Xanthobacter autotrophicus; Cupriavidus basilensis and Cupriavidus Examples of such hydrogen-producing bacteria include Cupriavidus bacteria such as Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus pauculus, and Cupriavidus taiwanensis; Hydrogenophaga bacteria such as Hydrogenophaga pseudoflava; and Variovorax bacteria such as Variovorax paradoxus. Analysis of the genomic DNA of these bacteria by the present inventors has revealed that all four genes constituting the biotin biosynthetic system, bioF, bioA, bioD, and bioB, are conserved on the chromosome, strongly suggesting that these bacteria are capable of biotin biosynthesis themselves (i.e., are not inherently biotin-requiring).

[0029] Examples of Hydrogenophilus bacteria include Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038.

[0030] Among these, hydrogenophilus bacteria are preferred, with hydrogenophilus thermorteolus being more preferred, due to their top-level growth rate and carbon dioxide fixation ability among carbon dioxide fixating microorganisms. Hydrogenophilus bacteria can be easily isolated from all over the world. A preferred strain of hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain. The hydrogenophilus thermorteolus TH-1 (NBRC 14978) strain exhibits the highest growth rate among carbon dioxide fixating microorganisms [Agricultural and Biological Chemistry, 41, 685-690 (1977)] (doubling in one hour). The hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.

[0031] Furthermore, hydrogen bacteria may be bacteria isolated from nature, or bacteria that have been genetically modified from bacteria isolated from nature. Modifications can be performed for purposes such as enabling high expression of introduced genes. Such modifications can be performed, for example, by removing (curing) endogenous plasmids in hydrogen bacteria. Methods for removing endogenous plasmids are well known, and include methods that utilize plasmid incompatibility, such as the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; destabilization by introducing a plasmid with the same replication origin as the endogenous plasmid; and destabilization by disrupting factors involved in the plasmid partition system.

[0032] Uses of Hydrogen Bacterial Mutants The hydrogen bacteria mutants of the present invention can produce substances by expressing genes originally present in their genomes. However, to produce target substances such as chemical compounds or recombinant proteins, they can also be introduced with metabolic enzyme genes encoding metabolic pathways necessary for the biosynthesis of target compounds or genes encoding target recombinant proteins. That is, the mutants of the present invention can have foreign metabolic enzyme genes encoding metabolic pathways necessary for the biosynthesis of target compounds or foreign genes encoding target recombinant proteins. The hydrogen bacteria mutants of the present invention can have one or more foreign genes. In the present invention, a gene introduced into a host is referred to as an "exogenous gene," whether it is an endogenous gene present in the genome of the host hydrogen bacteria or a heterologous gene not present in the genome of the host hydrogen bacteria.

[0033] The metabolic enzyme genes and recombinant protein genes introduced into the host hydrogen bacteria may be heterologous genes not present in the genome of the host hydrogen bacteria, or endogenous genes present in the genome of the host hydrogen bacteria, but heterologous genes are preferred. In the present invention, the term "protein" also includes polymers of 50 or fewer amino acids, commonly known as peptides.

[0034] These genes can be introduced into the hydrogen bacteria mutant using standard methods for introducing foreign genes into bacteria. These genes can be introduced directly into the hydrogen bacteria mutant, or they can be introduced into a transformation vector (e.g., a plasmid vector, a phage vector, a cosmid, a fosmid, a BAC, etc.) containing these genes and then introduced into the hydrogen bacteria mutant. The vector used for transformation may contain DNA capable of autonomous replication in the hydrogen bacterium. Examples include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6). Among these, pCAMO-6 (SEQ ID NO: 9) is preferred. pCAMO-6 can be prepared from its DNA sequence by those skilled in the art using gene synthesis services, etc. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and the OXB1, OXB11 to OXB20 promoters from Oxford Genetics, and examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage λt0 transcription terminator, the T7 terminator, etc. These genes can be introduced (transformed) into hydrogen bacteria mutants using conventional methods, such as the calcium chloride method, the rubidium chloride method, and the electric pulse method (electroporation).

[0035] (2) Method for Producing Target Substances The hydrogen bacteria mutant of the present invention, in which one or more of the biotin biosynthetic pathway components bioF, bioA, bioD, and bioB are disrupted, or which further contains an exogenous gene for producing (especially high-yielding) a target substance, can be cultured in a medium containing little or no biotin, thereby suppressing or arresting cell growth of the hydrogen bacteria mutant. Under this environment, little or no carbon dioxide is taken up and used for bacterial cell production during growth, allowing for efficient production of target substances such as compounds and proteins. Preferably, the hydrogen bacteria mutant containing the exogenous gene is grown in a medium containing biotin, and then cultured in a medium containing little or no biotin, thereby suppressing or arresting cell growth of the hydrogen bacteria mutant.

[0036] Because hydrogen bacteria can grow using hydrogen as an energy source and carbon dioxide as the sole carbon source, they can efficiently fix carbon dioxide by producing target substances using essentially only carbon dioxide as a carbon source (especially using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to culture the hydrogen bacteria mutant using essentially only carbon dioxide as a carbon source (especially using only carbon dioxide as a carbon source). In the present invention, "using carbon dioxide as the sole carbon source" encompasses the inevitable contamination with other carbon sources. For this reason, it is preferable to use an inorganic medium that does not contain organic matter, including inorganic carbon sources such as carbonates. Furthermore, because many organic matters contained in culture media generally contain biotin, inorganic media are also preferred. The pH of the culture medium used for cultivation is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within these ranges, bacterial growth and mixed gas solubility in the medium are high, allowing for highly efficient production of target substances.

[0037] When culturing a hydrogen bacteria mutant using substantially only carbon dioxide as a carbon source, the culturing may be carried out while supplying a gas containing carbon dioxide, preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide. The gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as the target substance can be efficiently produced.

[0038] For batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured statically or with shaking. Shaking culture is preferred because it improves the dissolution of the mixed gas into the medium. For continuous culture, the mixed gas can be continuously supplied to the culture vessel while being cultured with shaking, or the mixed gas can be introduced into the medium by bubbling while the transformant is cultured with stirring. The volume ratio of hydrogen, oxygen, and carbon dioxide in the supply gas (hydrogen:oxygen:carbon dioxide) is preferably 1.75-7.5:1:0.25-3, more preferably 5-7.5:1:1-2, and even more preferably 6.25-7.5:1:1.5. Within this range, the growth of the hydrogen bacteria mutant is favorable and the target substance can be efficiently produced. The supply rate of the mixed gas or raw material gas is 10-60 L / h, preferably 10-40 L / h, and more preferably 10-20 L / h per 1 L of medium. Within this range, the growth of the hydrogen bacteria mutant is favorable, the target substance can be produced efficiently, and waste of the mixed gas is reduced. The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and even more preferably 50 to 52°C. Within this range, the growth of the hydrogen bacteria mutant is favorable, and the target substance can be produced efficiently.

[0039] Because the mutants of the present invention are endowed with the trait of biotin auxotrophy, they can be cultured in a biotin-containing medium supplemented with biotin in the range of 50 to 200 μg / L for bacterial growth. The culture can be continued until the bacterial cell concentration, based on the volume of the medium, reaches, for example, 1 to 50% (w / v), preferably 2 to 20% (w / v). The grown hydrogen bacteria mutants can then be transferred to a biotin-deficient medium (a biotin concentration of 10 μg / L or less, preferably 1 μg / L or less, preferably 0.1 μg / L or less, and particularly a medium that is substantially free of or does not contain biotin) and cultured. Culture in the biotin-deficient medium can be continued until the desired amount of the target substance is achieved. The conditions for culture in the biotin-containing medium and the biotin-deficient medium, other than the biotin concentration, may be the same or different.

[0040] By culturing as described above, target substances such as compounds and proteins are produced in the culture medium, and the target substances can be recovered. In the case of many low molecular weight compounds, the target compounds can be recovered by recovering the culture medium. However, depending on the physical properties of the target compounds, the target compounds can also be separated from the culture medium by known methods. Such known methods include membrane separation and distillation.

[0041] When a protein is produced, secretory proteins are processed intracellularly and secreted extracellularly, and the recombinant protein can be recovered by recovering the culture medium. Non-secretory proteins can be separated and purified by known methods after collecting and disrupting the bacterial cells. Examples of such known methods include methods that utilize differences in solubility, such as salting out or solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, or gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0042] Methods for confirming the isolated and purified protein include, for example, Western blotting, activity measurement, etc. The structure of the purified protein can also be clarified by amino acid analysis, amino-terminal analysis, primary structure analysis, etc.

[0043] The present invention will now be described with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0044] (1) Preparation of a bioB-disrupted mutant of Hydrogenophilus thermorteolus Insertion of a counterselection marker into pCAMO-6 The gene disruption vector was constructed by inserting into pCAMO-6 the rpsL gene, which serves as a counterselection marker, and a DNA fragment containing sequences adjacent to the bioB gene of the genomic DNA of Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) (sequences homologous to the 5'-flanking region of the bioB gene and sequences homologous to the 3'-flanking region of the bioB gene).

[0045] Genomic DNA was extracted from a wild-type TH-1 strain (a streptomycin-sensitive strain) using standard methods. Using the extracted genomic DNA as a template, a DNA fragment containing the rpsL gene, which encodes the S12 ribosomal protein and is responsible for streptomycin sensitivity, was amplified by PCR. The following primers were used for PCR. PCR was performed using a Life Technologies DNA Thermal Cycler and KOD One PCR Master Mix (Toyobo Co., Ltd.) as the reaction reagent. Primers for amplifying the wild-type rpsL gene of the TH-1 strain: (a-1) 5'-CTGGAGGAGAAACGCATATGCCAACCATCAACCAGTTGGTG-3' (SEQ ID NO: 10) (b-1) 5'-CGACGGAGCTCGAATTCTTATTTCTTGCCCGCAGCGGC-3' (SEQ ID NO: 11). Primers (a-1) and (b-1) contained sequences homologous to the pCAMO-6 vector. The resulting reaction mixture was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 0.4 kbp was detected. The agarose gel portion containing the DNA fragment corresponding to the rpsL gene was excised from the agarose gel, and the DNA fragment of the rpsL gene was recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0046] To perform DNA ligation by Gibson Assembly, the plasmid vector pCAMO-6 (SEQ ID NO: 9) was amplified by PCR using the DNA consisting of the base sequence of SEQ ID NO: 9 as a template and the following primers. Primers used to amplify the plasmid vector pCAMO-6: (a-2) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 12) (b-2) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 13). The resulting reaction solution was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 5.2 kbp was detected. The agarose gel portion containing the DNA fragment corresponding to the vector was excised from the agarose gel, and the DNA fragment was recovered.

[0047] The DNA fragment of the pCAMO-6 vector synthesized above and the DNA fragment of the rpsL gene were ligated using Gibson Assembly Master Mix (New England Biolabs). The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method. The resulting transformants were plated onto LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The sequence of the rpsL gene inserted into each plasmid was analyzed by Sanger analysis at Eurofins Genomics, and confirmed to match the sequence in the database. The resulting vector was named pTCL3. pTCL3 contains the streptomycin sensitivity gene (rpsL gene) and the kanamycin resistance gene derived from pCAMO-6.

[0048] Obtaining a streptomycin-resistant strain Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) (hereinafter referred to as "TH-1 strain") was cultured in liquid medium A [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, and CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The bacteria were inoculated into a glass vial containing 5 mL of TH-1000 (7.0) and incubated at 52°C under a gas mixture of H2:O2:CO2 (7.5:1:1.5) with a platinum loop. After 24 hours, the culture was inoculated onto LB solid medium containing 100 μg / ml streptomycin and incubated at 52°C for 72 hours. Three colonies were confirmed on the LB solid medium containing 100 μg / ml streptomycin. These colonies were then inoculated into a glass vial containing 5 mL of liquid medium A containing 100 μg / ml streptomycin with a platinum loop and incubated at 52°C under a gas mixture of H2:O2:CO2 (7.5:1:1.5). Since bacterial growth was observed after 24 hours, these strains were streptomycin-resistant variants of the TH-1 strain, and one of them was designated strain SR88. Streptomycin-resistant strains of hydrogen bacteria emerge at a certain rate when a culture of hydrogen bacteria is cultured on a solid medium containing 10 to 100 μg / ml of streptomycin.

[0049] Preparation of bioB gene (HPTL_1304) disruption vector: A disruption vector was constructed by inserting the DNA sequence near the bioB gene into the pTCL3 vector. Using the genomic DNA of the TH-1 strain as a template, the region containing a sequence homologous to the 5'-flanking region of the bioB gene (hereinafter sometimes abbreviated as the "5' homologous region") and the region containing a sequence homologous to the 3'-flanking region of the bioB gene (hereinafter sometimes abbreviated as the "3' homologous region") were amplified using the following primers: Primers used for amplification of the 5' homologous region of the bioB gene: (a-3) 5'-GAATCGCGAAATAAGGCCTCACGAAACGCGGTGGCATGCC-3' (SEQ ID NO: 14) (b-3) 5'-CGCATTCTCCAGAGCAAGGAAGCGTTG-3' (SEQ ID NO: 15) Primers used for amplification of the 3' homologous region of the bioB gene: (a-4) 5'-TCCTTGCTCTGGAGAATGCGCCCGGACGTCGCGCACGACAACG-3' (SEQ ID NO: 16) (b-4) 5'-CTATCAACAGGAGTCCTCACGTTCCGTTCCTGTGCGGTGGTG-3' (SEQ ID NO: 17) Primers (a-3) and (b-4) contain sequences homologous to the vector pTCL3, and the sequences of the 20 bases on the 5' side of primers (a-4) and (b-3) are complementary to each other. The resulting reaction mixture was subjected to agarose gel electrophoresis, which detected DNA fragments of approximately 1.4 kbp for the 5' homologous region and approximately 2.6 kbp for the 3' homologous region. The agarose gel sections containing these DNA fragments were excised from the agarose gel, and the DNA fragments were recovered.

[0050] To perform DNA ligation using Gibson Assembly, the plasmid vector pTCL3 was amplified by PCR. The following primers were used for PCR: Primers for amplification of plasmid vector pTCL3: (a-5) 5'-GGACTCCTGTTGATAGATCCAGTAATGACC -3' (SEQ ID NO: 18) (b-5) 5'-GGCCTTATTTCGCGATTCCCAAGAAGACAG -3' (SEQ ID NO: 19) The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 4.5 kbp was detected. The agarose gel portion containing this vector DNA fragment was excised from the agarose gel, and the vector DNA fragment was recovered.

[0051] The DNA fragments of the pTCL3 vector synthesized above were ligated to the 5' homologous region and the 3' homologous region DNA fragments using Gibson Assembly Master Mix to form circular DNA. The resulting reaction mixture was transformed into Escherichia coli JM109 by heat shock, and the resulting transformants were plated onto LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The plasmid sequence was analyzed by Sanger analysis and confirmed to match the sequence in the database. The resulting plasmid was named pTCL3-bioB. pTCL3-bioB contains both flanking regions of the bioB gene (HPTL_1304), the streptomycin sensitivity gene (rpsL gene), and the kanamycin resistance gene derived from pCAMO-6.

[0052] To prepare circular DNA (pTCL3-bioB-Δori) from pTCL3-bioB by removing the origin of replication sequence, a PCR reaction was performed using the following primers. Primers for amplifying the plasmid vector pTCL3-bioB: (a-6) 5'-GGCGGAGCCTATGGAAAAACGCC -3' (SEQ ID NO: 20) (b-6) 5'-CGGGGGTCTGACGCTCAGTGGAAC -3' (SEQ ID NO: 21). The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 8.5 kbp was detected. The agarose gel portion containing this vector DNA fragment was excised from the agarose gel, and the vector DNA fragment was recovered.

[0053] The recovered DNA fragment was added with T4 ligase and T4 polynucleotide kinase (Takara Bio) and incubated at 37°C for 1 hour to circularize the DNA, yielding pTCL3-bioB-Δori. This was used to transform streptomycin-resistant SR88 strains by electroporation, and the transformants were plated on LB solid medium containing 50 μg / mL kanamycin but no streptomycin and cultured at 52°C for 24 hours. If recombination occurs between the 5' or 3' homologous region of the circular DNA and the 5' or 3' homologous region of the SR88 genomic DNA, the circular DNA is incorporated into the genomic DNA, enabling growth via the kanamycin resistance gene contained in the circular DNA. If recombination does not occur, the circular DNA without the replication origin is lost during growth, allowing recombinant strains to be selected using kanamycin-containing medium. The first round of homologous recombination involves a single crossover, resulting in the entire introduced circular DNA, pTCL3-bioB-Δori, being integrated into the SR88 genome.

[0054] A small amount of bacterial cells was collected from colonies grown on kanamycin-containing plates and subjected to colony PCR using primers (a-3) and (b-4). Colonies that amplified both a 5.0-kbp DNA fragment derived from the SR88 genomic DNA (including the bioB gene and its flanking regions) and a 4.0-kbp DNA fragment derived from pTCL3-bioB-Δori, which lacks the bioB gene, were selected and inoculated into 5 mL of liquid medium A containing 50 μg / mL kanamycin in a glass vial using a platinum loop. Cultures were then incubated at 52°C for 24 hours with shaking under a gas mixture of H2:O2:CO2 (7.5:1:1.5).

[0055] The resulting culture was inoculated onto LB solid medium containing 100 μg / mL streptomycin and cultured at 52°C for 24 hours. A second homologous recombination occurred within the genomic DNA. When a second homologous recombination occurred between the 3' region derived from pTCL3-bioB-Δori and the 3' region derived from the SR88 genome, the bioB gene, kanamycin resistance gene, and rpsL gene were lost from the SR88 genomic DNA, and circular DNA (containing the bioB gene and its flanking regions, the kanamycin resistance gene, and the rpsL gene) was excised. This resulted in the generation of a SR88 strain lacking the bioB gene. Because the excised circular DNA lacked a replication origin, it was lost during bacterial growth. Furthermore, bacteria that did not undergo a second homologous recombination were sensitive to streptomycin due to the rpsL gene and could not grow on this medium. Furthermore, when a second homologous recombination occurs between the 5' region derived from pTCL3-bioB-Δori and the 5' region derived from the genome of the SR88 strain, pTCL3-bioB-Δori is excised, returning the strain to SR88. Therefore, the strains that grow on streptomycin-containing LB solid medium are SR88 strains with the bioB gene deleted and SR88 strains.

[0056] A small amount of bacterial cells was collected from colonies grown on streptomycin-containing LB solid medium and subjected to colony PCR using primers (a-3) and (b-4). The colony in which the 5.0-kbp DNA fragment (containing the bioB gene) derived from wild-type genomic DNA was not detected and only the 4.0-kbp DNA fragment derived from DNA lacking the bioB gene was amplified was a disruption mutant of the SR88 strain in which the bioB gene in the genome had been deleted, and this strain was named Del_bioB.

[0057] Confirmation of biotin auxotrophy of the bioB-disrupted mutant. LB solid medium contains a significant amount of biotin derived from organic substances such as yeast extract, which is thought to enable growth of biotin-auxotrophic strains. Therefore, the biotin auxotrophy of the Del_bioB strain was confirmed by culturing it in liquid medium A, which consists only of inorganic salts. Colonies of the Del_bioB strain were cultured in liquid medium A containing 5 μg / L biotin, centrifuged, and washed by removing the supernatant. The cells were then inoculated into biotin-free medium and medium containing 5 μg / L biotin, respectively, and cultured at 52°C for 24 hours. Growth was observed even in the biotin-free medium. This is thought to be due to the small amount of biotin required for growth, and the residual biotin remaining during washing supported growth. However, when the culture was further inoculated into biotin-free medium and cultured for an additional 24 hours, the bacterial density was approximately 1.3% of that of the wild-type TH-1 strain cultured for 24 hours, and bacterial growth was strongly suppressed. Furthermore, the expected induction of autolysis and bacteriolysis was not observed. This confirmed that disruption of the bioB gene conferred biotin auxotrophy. Furthermore, consumption of the enclosed gas mixture was observed even during growth suppression, and growth resumed rapidly upon reintroduction of biotin, confirming that major intracellular metabolic functions, including carbon dioxide uptake, were maintained even under biotin-limited conditions.

[0058] (2) Substance production using a bioB-disrupted mutant of Hydrogenophilus thermorteolus. An expression vector carrying a gene for a metabolic enzyme required for the biosynthesis of the compound to be produced or a gene encoding the protein to be produced is introduced into the resulting bioB-disrupted mutant, Del_bioB, using electroporation, and the strain is grown in a biotin-containing medium. The resulting cells are thoroughly washed and then cultured in a biotin-free medium, resulting in more efficient substance production than conventional cultures using wild-type strains.

[0059] The base sequences of SEQ ID NOs: 1 to 9 are shown below.

[0060] The mutants of the present invention can produce target substances such as compounds and proteins with high efficiency using carbon dioxide as the sole carbon source, and therefore can solve the problem of global warming caused by increased carbon dioxide while industrially producing food, feed, fuel, chemical products, pharmaceuticals, or the raw materials for these with high efficiency.

Claims

1. A mutant of hydrogen bacteria in which one or more of the following genes (1) to (4) present on the chromosome of hydrogen bacteria have been disrupted: (1) the 8-amino-7-oxononanoic acid synthase gene, (2) the 8-amino-7-oxononanoic acid aminotransferase gene, (3) the desthiobiotin synthase gene, and (4) the biotin synthase gene.

2. The bioF of the hydrogen bacterium is any one of the following genes (a1) to (a5): (a1) DNA comprising the nucleotide sequence of SEQ ID NO: 1; (a2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 1, and encoding a polypeptide having 8-amino-7-oxononanoic acid synthase activity to produce 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA; (a3) ​​DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (a4) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 2, and having 8-amino-7-oxononanoic acid synthase activity to produce 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA; (a5) DNA comprising an amino acid sequence in which 1 to 40 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, and encoding a polypeptide having 8-amino-7-oxononanoic acid synthase activity to produce 8-amino-7-oxononanoic acid from pimeloyl ACP / CoA. The bioA gene of hydrogen bacteria is any one of the genes (b1) to (b5) below: (b1) DNA comprising the nucleotide sequence of SEQ ID NO: 3; (b2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 3, and encoding a polypeptide having 8-amino-7-oxononanoic acid aminotransferase activity to produce 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid; (b3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4; (b4) DNA encoding a polypeptide comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 4, and having 8-amino-7-oxononanoic acid aminotransferase activity to produce 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid; (b5) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 40 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4, and having 8-amino-7-oxononanoic acid aminotransferase activity that produces 7,8-diaminononanoic acid from 8-amino-7-oxononanoic acid. The bioD gene of the hydrogen bacterium is any one of the genes (c1) to (c5) below:(c1) DNA comprising the nucleotide sequence of SEQ ID NO: 5; (c2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 5 and encoding a polypeptide having desthiobiotin synthase activity to produce desthiobiotin from 7,8-diaminononanoic acid; (c3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (c4) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 6 and having desthiobiotin synthase activity to produce desthiobiotin from 7,8-diaminononanoic acid; (c5) DNA encoding a polypeptide having an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6 and having desthiobiotin synthase activity to produce desthiobiotin from 7,8-diaminononanoic acid. The hydrogen bacterium mutant according to claim 1, wherein bioB of the hydrogen bacterium is any one of the genes (d1) to (d5) below. (d1) DNA comprising the nucleotide sequence of SEQ ID NO: 7; (d2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 7 and encoding a polypeptide having biotin synthase activity to synthesize biotin from desthiobiotin; (d3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; (d4) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 8 and having biotin synthase activity to synthesize biotin from desthiobiotin; (d5) DNA encoding a polypeptide having an amino acid sequence in which 1 to 30 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 8 and having biotin synthase activity to synthesize biotin from desthiobiotin; 3. A hydrogen bacterium mutant according to claim 1 or 2, in which the entire or partial region of any one or more of the 8-amino-7-oxononanoic acid synthase gene, the 8-amino-7-oxononanoic acid aminotransferase gene, the desthiobiotin synthase gene, and the biotin synthase gene has been deleted, or nucleotides have been inserted or added to the gene, or the entire or partial region of the gene has been replaced with other nucleotides, thereby disrupting the gene.

4. A hydrogen bacterium mutant according to any one of claims 1 to 3, which has an exogenous gene for producing a target substance.

5. The hydrogen bacterium mutant according to claim 4, wherein (i) the target substance is a compound and the exogenous gene is a metabolic enzyme gene required for the biosynthesis of the compound, or (ii) the target substance is a protein and the exogenous gene is a gene encoding the protein.

6. The hydrogen bacterium mutant according to any one of claims 1 to 5, wherein the hydrogen bacterium is a bacterium of the genus Hydrogenophilus.

7. A method for producing a target substance, comprising the step of culturing the hydrogen bacteria mutant according to claim 4 or 5 in a biotin-deficient medium.

8. The method according to claim 7, further comprising a step of culturing the hydrogen bacteria mutant of claim 4 or 5 in a medium containing biotin to grow it before the step of culturing it in a biotin-deficient medium.

9. The method according to claim 7 or 8, wherein the culture is carried out using carbon dioxide as substantially the only carbon source.

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