Genetic recombinant of hydrogenophilic bacteria with enhanced valine production ability

Abstract

A bacterium of the genus Hydrogenophilus, wherein a mutant acetolactate synthase III small subunit is formed, wherein the mutant acetolactate synthase III small subunit is contained in SEQ ID A mutant amino acid sequence having any one of the following amino acid substitutions (a) to (h) in the amino acid sequence of NO: 1: (a) Asn as the 11th amino acid is replaced by Asp, His or Ala; (b) Gly as the 14th amino acid is replaced by Asp or Ala; (c) Ser as the 17th amino acid is replaced by Phe; (d) Asn as the 29th amino acid is replaced by Lys, Tyr, Asp or His; (e) Thr as the 34th amino acid is replaced by Ile; (f) Ala as the 36th amino acid is replaced by Val; (g) Arg as the 84th amino acid and all subsequent amino acids are deleted; and (h) Leu as the 131st amino acid is replaced by Arg, and the hydrogenophilic bacterium is capable of effectively producing valine using carbon dioxide as the sole carbon source.

Description

Technical Field

[0001] The present invention relates to a recombinant Hydrogenophilus bacterium genetically enhanced to produce an increased amount of valine, a method for producing valine using the recombinant, and a method for producing the recombinant. Background Art

[0002] Using microorganisms to produce chemical products

[0003] The Paris Agreement, adopted in 2015, stipulates that global greenhouse gas emissions should be rapidly reduced. Under the agreement, Japan has set a target of reducing its emissions of greenhouse gases such as carbon dioxide and methane by 26% by 2030 compared to 2013 levels.

[0004] Worldwide, the production of most chemicals relies on petroleum resources, exacerbating the problem of increasing greenhouse gas emissions. Therefore, getting rid of dependence on petroleum is an ideal strategy for producing chemicals, and research and development of biorefining to produce green chemicals from biomass is being seriously carried out in various countries. However, saccharification of biomass used as raw material for microbial fermentation requires complicated processes in addition to high costs.

[0005] As part of research efforts to move away from petroleum dependence, gases such as carbon dioxide, methane, and carbon monoxide have attracted attention as more sustainable carbon sources, and technologies for producing valuable chemicals and biofuels using microorganisms that utilize these gases have become the subject of intense interest. In particular, carbon fixation of carbon dioxide, which contributes significantly to global warming, and efficient utilization of carbon dioxide are highly anticipated.

[0006] Valine production

[0007] Valine, one of the essential amino acids, is a substance that can be used as an ingredient of medicines, foods or cosmetics or as a livestock feed additive. Up to now, valine has been produced by fermentation or chemical hydrolysis of protein.

[0008] However, compared with the fermentative production of lactic acid or ethanol, the fermentative production of valine from renewable resources involves a very large number of metabolic reaction steps starting from the sugar serving as the raw material. Due to this and other reasons, such as the feedback inhibition of metabolic enzymes by the valine product, the resulting low productivity has always been an obstacle to the industrial-scale production of valine.

[0009] In microbial cells, valine is produced from pyruvate, which is an important intermediate metabolite in living cells, through 4 steps. The core process is as follows: acetolactate is produced from pyruvate by the catalytic action of acetolactate synthase; then 2,3-dihydroxyisovalerate is produced from acetolactate by the catalytic action of ketol-acid reductoisomerase; then 2-ketoisovalerate is produced from 2,3-dihydroxyisovalerate by the catalytic action of dihydroxyacid dehydratase; and finally, the amino group is transferred from glutamate to 2-ketoisovalerate by the catalytic action of branched-chain amino acid aminotransferase to produce valine.

[0010] For acetolactate synthase involved in the biosynthesis of valine in Escherichia coli, there are three known isozymes I, II and III. Acetolactate synthase II is resistant to feedback inhibition by valine, while acetolactate synthase I and acetolactate synthase III are sensitive to feedback inhibition by valine. Therefore, in order to produce valine using microorganisms, an acetolactate synthase resistant to feedback inhibition by valine produced at a high level is required.

[0011] The acetolactate synthase is composed of a large subunit and a small subunit. The large subunit has a catalytic function, while the small subunit retains a regulatory function. It is precisely because of the function of the small subunit that the enzymatic activity of acetolactate synthase is sensitive to feedback inhibition by valine.

[0012] Among the techniques for enhancing the productivity of valine in microorganisms, the techniques described in Patent Documents 1 and 2 present methods involving the use of a mutant acetolactate synthase desensitized to feedback inhibition by valine.

[0013] In Patent Document 1, there is disclosed a technique for producing valine, which involves the use of Escherichia coli in which a gene encoding the small subunit of acetolactate synthase I (ilvN gene) is mutated so as to desensitize feedback inhibition by valine.

[0014] In Patent Document 2, there is disclosed a technique for producing valine, which involves the use of Escherichia coli in which a gene encoding the small subunit of acetolactate synthase III (ilvH gene) is mutated so as to desensitize feedback inhibition by valine.

[0015] However, each of the above methods is a method for producing valine using sugar as a carbon raw material, and is different from a method for producing valine using carbon dioxide as a carbon raw material.

[0016] Modification of genes in the genome

[0017] For example, when the gene encoding the small subunit of acetolactate synthase I or III undergoes recombination, such as a mutation causing desensitization of feedback inhibition to valine, a marker gene is used as an indicator for selecting cells in which the recombination has successfully occurred. Antibiotic resistance genes and the like are widely used as marker genes. Cells into which the antibiotic resistance gene has been introduced become able to grow in the presence of antibiotics, and thus the antibiotic resistance gene is used for selection of cells into which the gene has been introduced, i.e., positive selection.

[0018] However, the kind of the marker gene is limited, therefore, when the modification of gene is carried out at multiple sites (locus) on the genome, for example, when deletion, insertion or replacement, the marker gene needs to be recycled. After each recombinant where the locus of interest is modified is selected as an indicator using a positive selection marker such as a drug resistance gene, when the cell from which the positive selection marker has been removed can be selected from the recombinant, the positive selection marker can be used again at another locus. For this purpose, a method for efficiently selecting the cell from which the positive selection marker has been removed is needed.

[0019] In the modification of genes on the genome, when a positive selection marker gene, such as a drug resistance gene, is inserted into the genome in order to select modified cells, the expression of the inserted positive selection marker gene generally affects the expression of genes in its vicinity. In this case, in order to eliminate the influence of the expression of the inserted positive selection marker gene, there is a need for efficiently selecting cells in which only the positive selection marker gene is deleted while maintaining the modified genotype.

[0020] For the selection of cells from which the positive selection marker has been removed as described above, i.e., counter-selection, it is appropriate to use a genetic marker that kills cells when expressed, i.e., a counter-selection marker. According to this method, by using the positive selection marker and the counter-selection marker linked to each other, cells that are not killed due to the removal of the counter-selection marker are selected, and thus cells from which the positive selection marker and the counter-selection marker have been removed can be selectively obtained.

[0021] In many bacteria including E. coli, the levansucrase gene (sacB) derived from Bacillus subtilis is widely used as a counter-selection marker. E. coli into which the sacB gene is introduced becomes unable to grow in the presence of sucrose. Utilizing this property, by selecting E. coli grown in a medium supplemented with sucrose, E. coli without the sacB gene can be selectively obtained.

[0022] In Escherichia coli and the like, there is also a known counter-selection method involving the use of a streptomycin-sensitive gene.

[0023] It is known that streptomycin-resistant strains appear with a certain frequency even for bacteria that were originally sensitive to streptomycin serving as an antibiotic. Such streptomycin-resistant strains have a mutation caused in a gene encoding ribosomal protein S12 (rpsL gene), and amino acid substitution in ribosomal protein S12 caused by the mutation serves as a cause of streptomycin resistance. In this case, the mutated rpsL gene (hereinafter sometimes referred to as "rpsL * ”) is called the streptomycin resistance gene.

[0024] When the wild-type (naturally occurring) rpsL gene is introduced into such a streptomycin-resistant strain, the host bacterium becomes sensitive to streptomycin. That is, the wild-type rpsL gene is more sensitive to streptomycin than the rpsL gene that is a mutant. * In this case, the rpsL gene is sometimes referred to as a streptomycin-sensitive gene.

[0025] That is, there is a known counter-selection method involving obtaining a streptomycin-resistant strain of Escherichia coli or the like using a streptomycin-resistant strain as a host and using a wild-type rpsL gene (streptomycin-sensitive gene) as a counter-selection marker (Non-Patent Document 1).

[0026] Citation List

[0027] Patent Literature

[0028] [Patent Document 1] JP2001-231584A

[0029] [Patent Document 2] JP2008-099668A

[0030] Non-patent literature

[0031] [Non-patent document 1] "Counterselectable markers: untapped tools for bacterial genetics and pathogenesis", Reyrat JM, Pelicic V, Gicquel B, Rappuoli R. Infect. Immun. (1998) 66: 4011-4017 Summary of the invention

[0032] Technical issues

[0033] The first object of the present invention is to provide a recombinant hydrogenophilus bacterium capable of efficiently producing valine using carbon dioxide as a sole carbon source, and a method for efficiently producing valine using the recombinant.

[0034] A second object of the present invention is to provide a method for efficiently producing a recombinant in a hydrogenophilic bacterium.

[0035] The inventors of the present invention have conducted studies to achieve the above-mentioned object and have obtained the following findings.

[0036] Techniques used to modify host genes

[0037] Hydrogenophilus bacteria is a hydrogen oxidizing bacteria that grows by utilizing hydrogen energy to produce organic matter from carbon dioxide. Generally, hydrogen oxidizing bacteria have an extremely low growth rate. However, hydrogenophilus bacteria have a high growth rate and have a carbon fixation capacity to carbon dioxide that is much higher than plants and photosynthetic bacteria. Hydrogenophilus bacteria produce valine, but the productivity is low due to the feedback inhibition of valine by metabolic enzymes. In order to provide the ability to produce valine on an industrial scale, the coding gene of the enzyme that catalyzes the reaction for producing valine needs to introduce a mutation that desensitizes feedback inhibition therein. However, there is no technology for modifying (e.g., deleting, replacing or adding (including inserting)) the gene on the genome of hydrogenophilus bacteria.

[0038] In this regard, the inventors of the present invention have intensively studied techniques for genetic modification in hydrogenophilic bacteria and have successfully performed recombination of hydrogenophilic bacteria, wherein a positive selection marker can be recycled by combining a streptomycin-resistant strain of hydrogenophilic bacteria with a wild-type rpsL gene (streptomycin-sensitive gene).

[0039] That is, the inventors of the present invention found that a streptomycin-resistant strain of a bacterium of the genus Hydrogenophilus can be isolated, and the rpsL gene of the bacterium of the genus Hydrogenophilus can serve as a streptomycin-sensitive gene for the streptomycin-resistant strain, and thus the rpsL gene can be used as a counter-selection marker.

[0040] To date, no positive selection markers are known for use in hydrogenophilic bacteria. The inventors of the present invention have found that the introduction of the hygromycin phosphotransferase gene into hydrogenophilic bacteria provides hygromycin resistance, and thus the gene can be used as a positive selection marker. The inventors have also found that the introduction of the neomycin / kanamycin phosphotransferase gene or the kanamycin nucleotidyltransferase gene into hydrogenophilic bacteria provides kanamycin resistance, and thus the gene can be used as a positive selection marker.

[0041] A known example of a hygromycin phosphotransferase gene is the gene encoding Escherichia coli hygromycin B phosphotransferase (hph) (Plasmid-encoded hygromycin B resistance: the sequence of hygromycinB phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae, Gritz L, Davies J. Gene (1983) 25: 179-188).

[0042] Known examples of genes for providing kanamycin resistance are the gene encoding the neomycin phosphotransferase of the Escherichia coli transposon Tn5 (nptII) and the gene encoding the kanamycin nucleotidyltransferase of Staphylococcus aureus (knt) (Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum, Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. Gene (1994) 145: 69-73; Comparative studies of the enzyme and nucleotide sequences of the kanamycin inactivating enzyme encoded by the plasmid from Thermobacterium thermophilum and by the plasmid pUB110). sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110), Matsumura M, Katakura Y, Imanaka T, Aiba S. J. Bacteriol. (1984) 160:413-420).

[0043] According to the studies conducted by the inventors of the present invention, it was found that although the introduction of a heterologous gene into a bacterium of the genus Hydrogenophilus does not cause its expression in many cases, the hygromycin phosphotransferase gene does express a protein that is functional in the bacterium of the genus Hydrogenophilus and thus provides hygromycin resistance thereto. It was also observed that the neomycin / kanamycin phosphotransferase gene and the kanamycin nucleotidyltransferase gene each express a protein that is functional in the bacterium of the genus Hydrogenophilus and thus provides kanamycin resistance thereto.

[0044] Valine production

[0045] The ilvH gene encoding the small subunit of acetolactate synthase III is present in the genome of hydrogenophilus bacteria. Acetolactate synthase III is subject to feedback inhibition by valine, and therefore an attempt was made in the present invention to introduce a mutation expected to desensitize feedback inhibition by valine into the gene encoding acetolactate synthase in the genome of hydrogenophilus bacteria.

[0046] As a result, a mutant acetolactate synthase III was found in which feedback inhibition by valine is desensitized, in which, in SEQ ID NO: 1 used as the amino acid sequence of the small subunit of acetolactate synthase III of a hydrogenophilic bacterium, Asn at the 11th amino acid is replaced by Asp, His or Ala, Gly at the 14th amino acid is replaced by Asp or Ala, Ser at the 17th amino acid is replaced by Phe, Asn at the 29th amino acid is replaced by Lys, Tyr, Asp or His, Thr at the 34th amino acid is replaced by Ile, Ala at the 36th amino acid is replaced by Val, Arg at the 84th amino acid and all subsequent amino acids are deleted, or Leu at the 131st amino acid is replaced by Arg. That is, the growth of wild-type hydrogenophilus bacteria is inhibited in the presence of 1 mM valine, but the growth of hydrogenophilus bacteria into which the above mutation is introduced into the gene encoding acetolactate synthase III is not inhibited even in the presence of high concentrations of valine, and valine is accumulated in the culture medium. This hydrogenophilus recombinant bacteria produces valine more efficiently using carbon dioxide as the sole carbon source.

[0047] The present invention was accomplished on the basis of the above findings, and provides the following hydrogenophilic bacteria recombinant, method for producing valine, method for producing recombinant, etc.

[0048] Item 1. A mutant acetolactate synthase III small subunit according to any one of (1) to (3) below:

[0049] (1) A mutant acetolactate synthase III small subunit, which is composed of a mutant amino acid sequence having any one of the following mutations (a) to (h) in the amino acid sequence shown in SEQ ID NO: 1:

[0050] (a) Asn at amino acid position 11 is replaced by Asp, His, or Ala;

[0051] (b) Gly at amino acid position 14 is replaced by Asp or Ala;

[0052] (c) Ser at amino acid position 17 was replaced by Phe;

[0053] (d) Asn at amino acid position 29 is replaced by Lys, Tyr, Asp or His;

[0054] (e) Thr at amino acid position 34 is replaced by Ile;

[0055] (f) Ala at amino acid position 36 is replaced by Val;

[0056] (g) Arg at amino acid position 84 and all subsequent amino acids are deleted; and

[0057] (h) Leu at amino acid position 131 was replaced by Arg;

[0058] (2) a mutant acetolactate synthase III small subunit, which is formed by an amino acid sequence having 90% or more identity with the mutant amino acid sequence described in (1), wherein the mutant acetolactate synthase III small subunit has acetolactate synthase III small subunit activity and suppresses feedback inhibition by valine,

[0059] The prerequisite is

[0060] When the mutant amino acid sequence has the mutation of (a), the amino acid at the 11th amino acid is Asp, His or Ala,

[0061] When the mutant amino acid sequence has the mutation of (b), the amino acid at the 14th amino acid is Asp or Ala,

[0062] When the mutant amino acid sequence has the mutation of (c), the amino acid at the 17th amino acid is Phe,

[0063] When the mutant amino acid sequence has the mutation of (d), the amino acid at the 29th amino acid is Lys, Tyr, Asp or His,

[0064] When the mutant amino acid sequence has the mutation of (e), the amino acid at the 34th amino acid is Ile,

[0065] When the mutant amino acid sequence has the mutation of (f), the amino acid at amino acid position 36 is Val,

[0066] When the mutant amino acid sequence has the mutation of (g), the amino acid at amino acid position 84 and all subsequent amino acids are deleted; and

[0067] When the mutant amino acid sequence has the mutation of (h), the amino acid at amino acid position 131 is Arg; and

[0068] (3) A mutant acetolactate synthase III small subunit, which is formed by an amino acid sequence having one or more amino acids deleted, substituted or added in the mutant amino acid sequence described in (1), wherein the mutant acetolactate synthase III small subunit has acetolactate synthase III small subunit activity and suppresses feedback inhibition by valine,

[0069] The prerequisite is

[0070] When the mutant amino acid sequence has the mutation of (a), the amino acid at the 11th amino acid is Asp, His or Ala,

[0071] When the mutant amino acid sequence has the mutation of (b), the amino acid at the 14th amino acid is Asp or Ala,

[0072] When the mutant amino acid sequence has the mutation of (c), the amino acid at the 17th amino acid is Phe,

[0073] When the mutant amino acid sequence has the mutation of (d), the amino acid at the 29th amino acid is Lys, Tyr, Asp or His,

[0074] When the mutant amino acid sequence has the mutation of (e), the amino acid at the 34th amino acid is Ile,

[0075] When the mutant amino acid sequence has the mutation of (f), the amino acid at amino acid position 36 is Val,

[0076] When the mutant amino acid sequence has the mutation of (g), the amino acid at amino acid position 84 and all subsequent amino acids are deleted; and

[0077] When the mutant amino acid sequence has the mutation of (h), the amino acid at amino acid position 131 is Arg.

[0078] Item 2. A mutant acetolactate synthase III small subunit gene according to any one of (4) to (6) below:

[0079] (4) A gene comprising a mutant base sequence having any one of the following mutations (i) to (p) in the base sequence shown in SEQ ID NO: 2:

[0080] (i) AAC at bases 31 to 33 is replaced by GAT, GAC, CAT, CAC, GCT, GCC, GCA, or GCG;

[0081] (j) GGC at bases 40 to 42 is replaced by GAT, GAC, GCT, GCC, GCA or GCG;

[0082] (k) TCG at bases 49 to 51 is replaced by TTT or TTC;

[0083] (l) AAC at bases 85 to 87 is replaced by AAA, AAG, TAT, TAC, GAT, GAC, CAT or CAC;

[0084] (m) ACC at bases 100 to 102 is replaced by ATT, ATC, or ATA;

[0085] (n) GCA at bases 106 to 108 is replaced by GTT, GTC, GTA or GTG;

[0086] (o) CGC at bases 250 to 252 is replaced by TAA, TAG or TGA; and

[0087] (p) CTC at bases 391 to 393 is replaced by CGT, CGC, CGA, CGG, AGA, or AGG;

[0088] (5) A gene consisting of a base sequence having 90% or higher identity with the mutant base sequence described in (4), the gene encoding a mutant acetolactate synthase III small subunit having acetolactate synthase III small subunit activity and suppressing feedback inhibition by valine,

[0089] The prerequisite is

[0090] When the mutant base sequence has the mutation of (i), the bases at bases 31 to 33 are GAT, GAC, CAT, CAC, GCT, GCC, GCA or GCG,

[0091] When the mutant base sequence has the mutation of (j), the bases at bases 40 to 42 are GAT, GAC, GCT, GCC, GCA or GCG,

[0092] When the mutant base sequence has a mutation of (k), the bases at bases 49 to 51 are TTT or TTC,

[0093] When the mutant base sequence has the mutation of (1), the bases at bases 85 to 87 are AAA, AAG, TAT, TAC, GAT, GAC, CAT or CAC,

[0094] When the mutant base sequence has a mutation of (m), the bases at bases 100 to 102 are ATT, ATC or ATA,

[0095] When the mutant base sequence has a mutation of (n), the bases at bases 106 to 108 are GTT, GTC, GTA or GTG,

[0096] When the mutant base sequence has the mutation of (o), the bases at bases 250 to 252 are TAA, TAG or TGA, and

[0097] When the mutant base sequence has the mutation of (p), the bases at bases 391 to 393 are CGT, CGC, CGA, CGG, AGA or AGG; and

[0098] (6) A gene which hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the mutant base sequence described in (4), and which encodes a mutant acetolactate synthase III small subunit having acetolactate synthase III small subunit activity and suppressing feedback inhibition by valine,

[0099] The prerequisite is

[0100] When the mutant base sequence has the mutation of (i), the bases at bases 31 to 33 are GAT, GAC, CAT, CAC, GCT, GCC, GCA or GCG,

[0101] When the mutant base sequence has the mutation of (j), the bases at bases 40 to 42 are GAT, GAC, GCT, GCC, GCA or GCG,

[0102] When the mutant base sequence has a mutation of (k), the bases at bases 49 to 51 are TTT or TTC,

[0103] When the mutant base sequence has the mutation of (1), the bases at bases 85 to 87 are AAA, AAG, TAT, TAC, GAT, GAC, CAT or CAC,

[0104] When the mutant base sequence has a mutation of (m), the bases at bases 100 to 102 are ATT, ATC or ATA,

[0105] When the mutant base sequence has a mutation of (n), the bases at bases 106 to 108 are GTT, GTC, GTA or GTG,

[0106] When the mutant base sequence has the mutation of (o), the bases at bases 250 to 252 are TAA, TAG or TGA, and

[0107] When the mutant base sequence has the mutation of (p), the bases at bases 391 to 393 are CGT, CGC, CGA, CGG, AGA or AGG.

[0108] Item 3. A bacterium of the genus Hydrogenophilus that produces the mutant acetolactate synthase III small subunit described in Item 1.

[0109] Item 4. A hydrogenophilus bacterium comprising the mutated acetolactate synthase III small subunit gene according to Item 2.

[0110] Item 5. A method for producing valine, comprising the step of culturing the hydrogenophilic bacterium described in Item 3 or 4 using carbon dioxide as essentially the only carbon source.

[0111] Item 6. A counter-selection marker for a bacterium of the genus Hydrogenophilus, comprising the streptomycin-sensitive gene of (7), (8) or (9) below:

[0112] (7) a streptomycin-sensitive gene formed by the base sequence shown in SEQ ID NO: 3;

[0113] (8) a gene consisting of a base sequence having 90% or higher identity with the base sequence shown in SEQ ID NO: 3, which encodes a polypeptide having an activity of providing streptomycin sensitivity to a streptomycin-resistant strain of a bacterium of the genus Hydrogenophilus; or

[0114] (9) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 3, the gene encoding a polypeptide having an activity of imparting streptomycin sensitivity to a streptomycin-resistant strain of a bacterium of the genus Hydrogenophilus.

[0115] Item 7. A method for producing a recombinant hydrogenophilus bacterium, the method comprising using the counter-selection marker described in Item 6 and a streptomycin-resistant strain of hydrogenophilus bacterium.

[0116] Item 8. A method for producing a recombinant hydrogenophilic bacterium, the method comprising the following steps:

[0117] (A) producing a marker DNA fragment having a marker cassette in which a DNA fragment containing a streptomycin-sensitive gene and a DNA fragment containing a gene for a positive selection marker are linked to each other, the marker cassette being inserted between the 5' terminal side region and the 3' terminal side region of a DNA fragment formed of a mutated target gene of a bacterium of the genus Hydrogenophilus;

[0118] (B) performing an operation of introducing the marker DNA fragment into a streptomycin-resistant strain of the hydrogenophilus bacterium, and then selecting each recombinant in which the target gene of the streptomycin-resistant strain has been replaced by the marker DNA fragment using the presence of the characteristic of the positive selection marker as an indicator; and

[0119] (C) introducing the DNA fragment formed by the mutated target gene of the bacterium of the genus Hydrogenophilus into each of the recombinants, and then selecting the recombinants in which the region of the marker DNA on the genome has been replaced by the mutated target gene using streptomycin resistance as an indicator.

[0120] Item 9. The method according to Item 8, wherein the streptomycin-sensitive gene is the gene of (7), (8) or (9) below:

[0121] (7) a streptomycin-sensitive gene formed by the base sequence shown in SEQ ID NO: 3;

[0122] (8) a gene consisting of a base sequence having 90% or higher identity with the base sequence shown in SEQ ID NO: 3, which encodes a polypeptide having an activity of providing streptomycin sensitivity to a streptomycin-resistant strain of the bacterium of the genus Hydrogenophilus; or

[0123] (9) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 3, the gene encoding a polypeptide having an activity of imparting streptomycin sensitivity to a streptomycin-resistant strain of the bacterium of the genus Hydrogenophilus.

[0124] Item 10. A method for producing a recombinant hydrogenophilic bacterium, the method comprising the following steps:

[0125] (D) producing a labeled circular DNA comprising a DNA fragment of a mutated target gene of a bacterium of the genus Hydrogenophilus and a labeling cassette, wherein a DNA fragment containing a streptomycin-sensitive gene and a DNA fragment containing a gene for a positive selection marker are linked to each other in the labeling cassette;

[0126] (E) performing an operation of introducing the marker circular DNA into a streptomycin-resistant strain of the hydrogenophilus bacterium, and then selecting each recombinant in which the marker circular DNA is linearized and inserted into the target gene of the streptomycin-resistant strain using the presence of the characteristic of the positive selection marker as an indicator;

[0127] (F) selecting each recombinant in which the region on the genome where the marker DNA is inserted has been eliminated from the recombinants using streptomycin resistance as an indicator; and

[0128] (G) selecting a recombinant having a mutation introduced into the target gene from the recombinants selected in step (F).

[0129] Item 11. The method according to Item 10, wherein the streptomycin-sensitive gene is the gene of (7), (8) or (9) below:

[0130] (7) a streptomycin-sensitive gene formed by the base sequence shown in SEQ ID NO: 3;

[0131] (8) a gene consisting of a base sequence having 90% or higher identity with the base sequence shown in SEQ ID NO: 3, which encodes a polypeptide having an activity of providing streptomycin sensitivity to a streptomycin-resistant strain of the bacterium of the genus Hydrogenophilus; or

[0132] (9) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 3, the gene encoding a polypeptide having an activity of imparting streptomycin sensitivity to a streptomycin-resistant strain of the bacterium of the genus Hydrogenophilus.

[0133] Item 12. A positive selection marker for a bacterium of the genus Hydrogenophilus, comprising the hygromycin resistance gene of (10), (11) or (12) below:

[0134] (10) a hygromycin resistance gene formed by the base sequence shown in SEQ ID NO: 4;

[0135] (11) a gene consisting of a base sequence having 90% or higher identity with the base sequence shown in SEQ ID NO: 4, which encodes a polypeptide having activity of providing hygromycin resistance to a bacterium of the genus Hydrogenophilus; or

[0136] (12) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 4, the gene encoding a polypeptide having an activity of imparting hygromycin resistance to a bacterium of the genus Hydrogenophilus.

[0137] Item 13. A positive selection marker for a bacterium of the genus Hydrogenophilus, comprising the kanamycin resistance gene of (13), (14) or (15) below:

[0138] (13) a kanamycin resistance gene formed by the base sequence shown in SEQ ID NO: 5 or 6;

[0139] (14) a gene consisting of a base sequence having 90% or higher identity with the base sequence shown in SEQ ID NO: 5 or 6, which encodes a polypeptide having activity of providing kanamycin resistance to a bacterium of the genus Hydrogenophilus; or

[0140] (15) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 5 or 6, the gene encoding a polypeptide having an activity of imparting kanamycin resistance to a bacterium of the genus Hydrogenophilus.

[0141] Beneficial effects

[0142] Measures to address the increase in atmospheric carbon dioxide require reducing carbon dioxide emissions and fixing the emitted carbon dioxide. In order to reduce carbon dioxide emissions, solar energy, wind energy, geothermal energy and similar energy sources are used instead of fossil energy. However, the use of such energy is not yet widespread enough to inhibit the accumulation of atmospheric carbon dioxide. Therefore, it is necessary to enhance the fixation of atmospheric carbon or the recycling of emitted carbon dioxide.

[0143] Carbon fixation of carbon dioxide can be done physically or chemically, but fixation using living cells can produce organic matter that can be used as food, feed and fuel. In this way, carbon dioxide itself becomes a resource that can be directly converted into valuable chemical products. Therefore, the dual problems of global warming caused by the increase of atmospheric carbon dioxide and the shortage of food, feed and fuel can be solved.

[0144] Hydrogen oxidizing bacteria can utilize the chemical energy produced by the reaction of hydrogen and oxygen and use carbon dioxide as the sole carbon source to grow. Since hydrogen oxidizing bacteria can produce chemical products from a mixture of oxygen, hydrogen and carbon dioxide gases as raw materials, the cells can efficiently assimilate carbon from carbon dioxide and be cultured in simple culture media. The growth of typical hydrogen oxidizing bacteria is usually slow, but the growth rate of hydrogenophilic bacteria is exceptionally high. The Journal of Mitsubishi Research Institute, No. 34, 1999, describes hydrogenophilic bacteria as follows: "Their proliferation capacity is so high that their carbon fixation capacity for carbon dioxide cannot be compared with that of plants, which truly demonstrates the high carbon dioxide fixation capacity of the microorganisms."

[0145] Hydrogenophilus bacteria produce valine in an amount required for survival, but not in an industrially applicable amount. The recombinant of the present invention has a mutation introduced into a gene related to valine production on the genome of the hydrogenophilus bacterium, and thus can produce valine with high efficiency.

[0146] As described above, among organisms having the ability to fix carbon dioxide, bacteria belonging to the genus Hydrogenophilus have particularly excellent carbon fixation ability for carbon dioxide. Therefore, the use of the recombinant of the present invention enables production of valine by fixing carbon from carbon dioxide on an industrial scale.

[0147] According to the present invention, hygromycin resistance gene and kanamycin resistance gene that can be used as positive selection marker in hydrogenophilic bacteria are provided.Streptomycin sensitive gene that can be used as counter-selection marker by being used together with streptomycin resistant strain of hydrogenophilic bacteria is also provided.Therefore, the road has been paved for the reorganization and genome modification of hydrogenophilic bacteria.Specifically, the discovery of streptomycin sensitive gene that can be used as counter-selection marker makes it possible to repeat the reorganization of hydrogenophilic bacteria, thereby allows its genome to be modified on a large scale.Described mark is not retained in the genome, and therefore, in the comparison of the recombinant strain of hydrogenophilic bacteria and its wild-type strain, can be discussed by directly correlating the difference between their phenotype, i.e., their characteristic and their genotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 is a schematic diagram illustrating an example of a method for producing a recombinant of a bacterium of the genus Hydrogenophilus using a streptomycin-sensitive gene as a counter-selection marker.

[0149] Figure 2 is a schematic diagram illustrating another example of a method for producing a recombinant of a bacterium of the genus Hydrogenophilus using a streptomycin-sensitive gene as a counter-selection marker. DETAILED DESCRIPTION

[0150] The present invention is described in detail below.

[0151] ( 1) Recombinant Hydrogenophilus bacteria

[0152] The recombinant bacterium of the genus Hydrogenophilus of the present invention produces a mutant acetolactate synthase III small subunit according to any one of the following (1) to (3).

[0153] (1) A mutant acetolactate synthase III small subunit, which is composed of a mutant amino acid sequence having any one of the following mutations (a) to (h) in the amino acid sequence shown in SEQ ID NO: 1:

[0154] (a) Asn at amino acid position 11 is replaced by Asp, His, or Ala;

[0155] (b) Gly at amino acid position 14 is replaced by Asp or Ala;

[0156] (c) Ser at amino acid position 17 was replaced by Phe;

[0157] (d) Asn at amino acid position 29 is replaced by Lys, Tyr, Asp or His;

[0158] (e) Thr at amino acid position 34 is replaced by Ile;

[0159] (f) Ala at amino acid position 36 is replaced by Val;

[0160] (g) Arg at amino acid position 84 and all subsequent amino acids are deleted; and

[0161] (h) Leu at amino acid position 131 was replaced by Arg.

[0162] SEQ ID NO: 1 shows the amino acid sequence of the naturally occurring acetolactate synthase III small subunit from Hydrogenophilus thermoluteolus.

[0163] (2) a mutant acetolactate synthase III small subunit, which is formed by an amino acid sequence having 90% or more, preferably 95% or more, even more preferably 98% or more, more preferably 99% or more identity with the mutant amino acid sequence described in (1), said mutant acetolactate synthase III small subunit having acetolactate synthase III small subunit activity and suppressing feedback inhibition by valine,

[0164] The prerequisite is

[0165] When the mutant amino acid sequence has the mutation of (a), the amino acid at the 11th amino acid is Asp, His or Ala,

[0166] When the mutant amino acid sequence has the mutation of (b), the amino acid at the 14th amino acid is Asp or Ala,

[0167] When the mutant amino acid sequence has the mutation of (c), the amino acid at the 17th amino acid is Phe,

[0168] When the mutant amino acid sequence has the mutation of (d), the amino acid at the 29th amino acid is Lys, Tyr, Asp or His,

[0169] When the mutant amino acid sequence has the mutation of (e), the amino acid at the 34th amino acid is Ile,

[0170] When the mutant amino acid sequence has the mutation of (f), the amino acid at amino acid position 36 is Val,

[0171] When the mutant amino acid sequence has the mutation of (g), the amino acid at amino acid position 84 and all subsequent amino acids are deleted; and

[0172] When the mutant amino acid sequence has the mutation of (h), the amino acid at amino acid position 131 is Arg.

[0173] In the present invention, the identity of the amino acid sequence is a value calculated using GENETYX version 17 (manufactured by Genetyx Corporation).

[0174] (3) A mutant acetolactate synthase III small subunit, which is formed by an amino acid sequence having one or more amino acids deleted, substituted or added in the mutant amino acid sequence described in (1), wherein the mutant acetolactate synthase III small subunit has acetolactate synthase III small subunit activity and suppresses feedback inhibition by valine,

[0175] The prerequisite is

[0176] When the mutant amino acid sequence has the mutation of (a), the amino acid at the 11th amino acid is Asp, His or Ala,

[0177] When the mutant amino acid sequence has the mutation of (b), the amino acid at the 14th amino acid is Asp or Ala,

[0178] When the mutant amino acid sequence has the mutation of (c), the amino acid at the 17th amino acid is Phe,

[0179] When the mutant amino acid sequence has the mutation of (d), the amino acid at the 29th amino acid is Lys, Tyr, Asp or His,

[0180] When the mutant amino acid sequence has the mutation of (e), the amino acid at the 34th amino acid is Ile,

[0181] When the mutant amino acid sequence has the mutation of (f), the amino acid at amino acid position 36 is Val,

[0182] When the mutant amino acid sequence has the mutation of (g), the amino acid at amino acid position 84 and all subsequent amino acids are deleted; and

[0183] When the mutant amino acid sequence has the mutation of (h), the amino acid at amino acid position 131 is Arg.

[0184] In the present invention, a plurality is, for example, 1 to 5, particularly 1 to 3, particularly 1 or 2, particularly 1.

[0185] Acetolactate synthase produces acetolactate from pyruvate. In the present invention, the fact that the polypeptide to be tested has acetolactate synthase III small subunit activity is confirmed by detecting the acetolactate synthase activity in the presence of acetolactate synthase III large subunit. Acetolactate synthase activity is measured by the following method: 100mM potassium phosphate (pH 7.5), 50mM sodium pyruvate, 10mM MgCl2, 0.1mM thiamine pyrophosphate, 0.1mM flavin adenine dinucleotide and the polypeptide to be tested are mixed, and the reduction of the absorption value (ε=17.5 / M·cm) at 333nm showing pyruvate absorption is used as an indicator.

[0186] In the present invention, the fact that the polypeptide to be tested suppresses the feedback inhibition of valine is confirmed by detecting the acetolactate synthase activity in the presence of valine. When the acetolactate synthase activity of the polypeptide to be tested is increased, even slightly, compared with the wild-type (naturally occurring) acetolactate synthase III in the presence of a certain concentration of valine and in the coexistence with the large subunit of acetolactate synthase III, it can be judged that the feedback inhibition of valine is suppressed.

[0187] An example of the recombinant hydrogenophilus bacterium of the present invention is a recombinant hydrogenophilus bacterium having an acetolactate synthase III small subunit gene having any one of the mutations (4) to (6) described below on its genome.

[0188] A specific example thereof is a recombinant of a bacterium of the genus Hydrogenophilus in which the acetolactate synthase III small subunit gene naturally present on the genome has been replaced with a mutant acetolactate synthase III small subunit gene of any one of (4) to (6) described below.

[0189] (4) A gene comprising a mutant base sequence having any one of the following mutations (i) to (p) in the base sequence shown in SEQ ID NO: 2:

[0190] (i) AAC at bases 31 to 33 is replaced by GAT, GAC, CAT, CAC, GCT, GCC, GCA, or GCG;

[0191] (j) GGC at bases 40 to 42 is replaced by GAT, GAC, GCT, GCC, GCA or GCG;

[0192] (k) TCG at bases 49 to 51 is replaced by TTT or TTC;

[0193] (l) AAC at bases 85 to 87 is replaced by AAA, AAG, TAT, TAC, GAT, GAC, CAT or CAC;

[0194] (m) ACC at bases 100 to 102 is replaced by ATT, ATC, or ATA;

[0195] (n) GCA at bases 106 to 108 is replaced by GTT, GTC, GTA or GTG;

[0196] (o) CGC at bases 250 to 252 is replaced by TAA, TAG or TGA; and

[0197] (p) CTC at bases 391 to 393 is replaced by CGT, CGC, CGA, CGG, AGA, or AGG.

[0198] SEQ ID NO: 2 shows the base sequence of the naturally occurring acetolactate synthase III small subunit gene of Hydrogenophilus thermoflavonoids.

[0199] (5) A gene consisting of a base sequence having 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity, more preferably 99% or more identity to the mutant base sequence described in (4), the gene encoding a mutant acetolactate synthase III small subunit having acetolactate synthase III small subunit activity and suppressing feedback inhibition by valine,

[0200] The prerequisite is

[0201] When the mutant base sequence has the mutation of (i), the bases at bases 31 to 33 are GAT, GAC, CAT, CAC, GCT, GCC, GCA or GCG,

[0202] When the mutant base sequence has the mutation of (j), the bases at bases 40 to 42 are GAT, GAC, GCT, GCC, GCA or GCG,

[0203] When the mutant base sequence has a mutation of (k), the bases at bases 49 to 51 are TTT or TTC,

[0204] When the mutant base sequence has the mutation of (1), the bases at bases 85 to 87 are AAA, AAG, TAT, TAC, GAT, GAC, CAT or CAC,

[0205] When the mutant base sequence has a mutation of (m), the bases at bases 100 to 102 are ATT, ATC or ATA,

[0206] When the mutant base sequence has a mutation of (n), the bases at bases 106 to 108 are GTT, GTC, GTA or GTG,

[0207] When the mutant base sequence has the mutation of (o), the bases at bases 250 to 252 are TAA, TAG or TGA, and

[0208] When the mutant base sequence has the mutation of (p), the bases at bases 391 to 393 are CGT, CGC, CGA, CGG, AGA or AGG.

[0209] In the present invention, the base sequence identity is a value calculated using GENETYX version 17 (manufactured by Genetyx Corporation).

[0210] (6) A gene which hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the mutant base sequence described in (4), and which encodes a mutant acetolactate synthase III small subunit having acetolactate synthase III small subunit activity and suppressing feedback inhibition by valine,

[0211] The prerequisite is

[0212] When the mutant base sequence has the mutation of (i), the bases at bases 31 to 33 are GAT, GAC, CAT, CAC, GCT, GCC, GCA or GCG,

[0213] When the mutant base sequence has the mutation of (j), the bases at bases 40 to 42 are GAT, GAC, GCT, GCC, GCA or GCG,

[0214] When the mutant base sequence has a mutation of (k), the bases at bases 49 to 51 are TTT or TTC,

[0215] When the mutant base sequence has the mutation of (1), the bases at bases 85 to 87 are AAA, AAG, TAT, TAC, GAT, GAC, CAT or CAC,

[0216] When the mutant base sequence has a mutation of (m), the bases at bases 100 to 102 are ATT, ATC or ATA,

[0217] When the mutant base sequence has a mutation of (n), the bases at bases 106 to 108 are GTT, GTC, GTA or GTG,

[0218] When the mutant base sequence has the mutation of (o), the bases at bases 250 to 252 are TAA, TAG or TGA, and

[0219] When the mutant base sequence has the mutation of (p), the bases at bases 391 to 393 are CGT, CGC, CGA, CGG, AGA or AGG.

[0220] In the present invention, "stringent conditions" means hybridization using a 6xSSC solution at a temperature of 50 to 60°C for 16 hours, followed by washing with a 0.1xSSC solution.

[0221] Examples of hydrogenophilus bacteria include Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, and Hydrogenophilus sp. Mar3. Specifically, Hydrogenophilus halorhabdus is preferred because its superior growth rate enables it to achieve the highest level of carbon fixation for carbon dioxide among microorganisms that fix carbon dioxide.

[0222] Hydrogenophilus bacteria have been readily isolated from a wide variety of regions around the world. A preferred strain of thermoyellow hydrogenophilus is strain TH-1 (NBRC 14978). Among microorganisms that fix carbon dioxide, thermoyellow hydrogenophilus strain TH-1 (NBRC 14978) exhibits a relatively fast growth rate (Agricultural and Biological Chemistry, 41, 685-690 (1977)). Internationally, thermoyellow hydrogenophilus strain NBRC 14978 is deposited under the Budapest Convention and is therefore available to the general public.

[0223] ( 2) Method for producing valine

[0224] In order to produce valine using the above-mentioned hydrogenophilus bacterial recombinant, the recombinant can be cultured in an inorganic defined or organic complex medium while supplying a mixed gas containing hydrogen, oxygen and carbon dioxide.

[0225] The supplied gas is preferably a mixture of hydrogen, oxygen and carbon dioxide. However, different kinds of gases may be mixed therein to such an extent that lactic acid can be efficiently produced.

[0226] Hydrogenophilus bacteria can use hydrogen as the energy source and use carbon dioxide to grow as the sole carbon source, therefore, by basically only using carbon dioxide (particularly by only using carbon dioxide) to produce the above-mentioned compound as the carbon source, the carbon in the carbon dioxide can be assimilated efficiently.Therefore, using inorganic culture medium that does not contain carbon source such as organic matter and carbonate, namely basically only using carbon dioxide (particularly only using carbon dioxide) to cultivate as the carbon source, is preferred. "Basically only using carbon dioxide as the carbon source" has covered the situation that other carbon sources of inevitable amount are mixed therein.In addition, the culture medium containing organic matter such as sugar, organic acid and amino acid and carbonate can also be used, and carbon dioxide is not supplied.

[0227] The pH of the culture medium is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. When the pH is within this range, bacteria grow well and mixed gases are well dissolved in the culture medium, and valine can be produced more efficiently.

[0228] When batch culture is used, the mixed gas can be trapped in an airtight culture container, and static culture or shaking culture can be performed. When continuous culture is used, the mixed gas can be continuously supplied to the airtight culture container and shaking culture can be performed, or the transformant can be cultured by introducing the mixed gas into the culture medium using an airtight culture container and bubbling at the same time. Shaking culture is preferred because better dissolution of the mixed gas in the culture medium can be obtained.

[0229] The volume ratio of hydrogen, oxygen and carbon dioxide (hydrogen: oxygen: carbon dioxide) in the supplied gas mixture is preferably 1.75 to 7.5: 1: 0.25 to 3, more preferably 5 to 7.5: 1: 1 to 2, and even more preferably 6.25 to 7.5: 1: 1.5. When the volume ratio is within this range, bacteria grow well and can efficiently produce the target compound.

[0230] The supply rate of the mixed gas or raw gas can be 10.5 to 60 L / hour, particularly 10.5 to 40 L / hour, particularly 10.5 to 21 L / hour per 1 L of culture medium. When the supply rate is within this range, the transformant grows well, valine can be efficiently produced, and the volume of unused mixed gas can be minimized.

[0231] The culture temperature is preferably 35 to 55° C., more preferably 37 to 52° C., more preferably 50 to 52° C. When the temperature is within this range, the transformant grows well, and valine can be produced efficiently.

[0232] By cultivating in the above manner, the target compound valine is produced in the reaction solution. Collecting the reaction solution allows the recovery of valine. However, valine can also be separated from the reaction solution by using known methods. Such known methods include ion exchange resins, concentration, crystallization, and the use of activated carbon adsorption and elution.

[0233] ( 3) Method for producing recombinant hydrogenophilic bacteria

[0234] The gene on the chromosome can be replaced with a mutant gene by the following method: the mutant gene is produced by PCR, the DNA containing the mutant gene is transformed into bacteria, and homologous recombination is produced between the mutant gene and the gene on the genome. In other words, mutations can be introduced into the gene on the chromosome. The enzyme protein encoded by the mutant gene has a three-dimensional structure different from the wild-type enzyme protein, causing functional changes.

[0235] As described above, the use of homologous recombination to modify genes on the genome is unprecedented in hydrogenophilic bacteria. In the present invention, a counter-selection method involving the use of streptomycin-resistant strains and streptomycin-sensitive genes in hydrogenophilic bacteria was developed, and genetic modification was performed based on the principle of site-specific incorporation of markers by homologous recombination and elimination of the markers by a second homologous recombination.

[0236] In this method, the streptomycin-sensitive gene described in (7), (8) or (9) below is used as a counter-selection marker. Those streptomycin-sensitive genes are functional in bacteria belonging to the genus Hydrogenophilus.

[0237] (7) a streptomycin-sensitive gene formed by the base sequence shown in SEQ ID NO: 3;

[0238] (8) a gene consisting of a base sequence having 90% or more, preferably 95% or more, even more preferably 98% or more, more preferably 99% or more identity with the base sequence shown in SEQ ID NO: 3, which encodes a polypeptide having an activity of providing streptomycin sensitivity to a streptomycin-resistant strain of a bacterium of the genus Hydrogenophilus; or

[0239] (9) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 3, the gene encoding a polypeptide having an activity of imparting streptomycin sensitivity to a streptomycin-resistant strain of a bacterium of the genus Hydrogenophilus.

[0240] Examples of the streptomycin-sensitive gene of (8) and (9) include the following genes (r), (s) and (t):

[0241] (r) a streptomycin-sensitive gene formed by a base sequence in which AAA at bases 127 to 129 in SEQ ID NO: 3 is replaced by CGT, CGC, CGA, CGG, AGA, AGG, AAT, AAC, ACT, ACC, ACA, ACG, ATT, ATC or ATA;

[0242] (s) a streptomycin-sensitive gene formed by a base sequence in which AAA at bases 262 to 264 in SEQ ID NO: 3 is replaced by CGT, CGC, CGA, CGG, AGA, AGG, GAA or GAG; and

[0243] (t) A streptomycin-sensitive gene formed by a base sequence in which CCG at bases 271 to 273 in SEQ ID NO: 3 is replaced by TTA, TTG, CTT, CTC, CTA, CTG, CAA or CAG.

[0244] As preferred promoters for causing the expression of the marker gene in hydrogenophilic bacteria, examples are provided: tac promoter, lac promoter, trc promoter and each of the promoters OXB1 and OXB11 to OXB20 from Oxford Genetics Ltd. As preferred terminators, examples are provided: the T1T2 terminator of the rRNA operon rrnB of Escherichia coli and the t0 transcription terminator of bacteriophage λ.

[0245] The fact that the gene to be tested is a gene encoding a polypeptide having an activity of providing streptomycin sensitivity to a streptomycin-resistant strain of a hydrogenophilic bacterium is confirmed by the fact that the growth of the streptomycin-resistant strain into which the gene to be tested is introduced is inhibited to the same extent as that of the wild-type hydrogenophilic bacterium in the presence of streptomycin (50 μg / mL or more).

[0246] An example of a method for producing a recombinant bacterial strain of the genus Hydrogenophilus using the above-mentioned counter-selection marker is a method comprising the following steps (A) to (C) (a first method, Figure 1 ):

[0247] (A) producing a marker DNA fragment having a marker cassette in which a DNA fragment containing a streptomycin-sensitive gene and a DNA fragment containing a gene for a positive selection marker are linked to each other, the marker cassette being inserted between the 5' terminal side region and the 3' terminal side region of a DNA fragment formed of a mutated target gene of a bacterium of the genus Hydrogenophilus;

[0248] (B) performing an operation of introducing the marker DNA fragment into a streptomycin-resistant strain of the hydrogenophilus bacterium, and then selecting each recombinant in which the target gene of the streptomycin-resistant strain has been replaced by the marker DNA fragment using the presence of the characteristic of the positive selection marker as an indicator; and

[0249] (C) introducing the DNA fragment formed by the mutated target gene of the bacterium of the genus Hydrogenophilus into each of the recombinants, and then selecting the recombinants in which the region of the marker DNA on the genome has been replaced by the mutated target gene using streptomycin resistance as an indicator.

[0250] ( A) Generation of labeled DNA fragments

[0251] Site-directed mutagenesis methods for introducing a mutation of interest (e.g., replacement, deletion, or addition (including insertion)) into a target gene are well known. For example, a target gene of a hydrogenophilic bacterium into which a mutation is introduced can be obtained by using a primer set in which one of the primers introduces the mutation of interest, and performing PCR using the genomic DNA of the hydrogenophilic bacterium as a template.

[0252] In this case, the following strategy is suitable: dividing the target gene into two parts, ie, the 5' terminal side region and the 3' terminal side region; amplifying the regions separately by PCR; and introducing one or more mutations into either or both of the regions.

[0253] Where appropriate, the amplified 5' terminal side region and 3' terminal side region are ligated to a vector that can be used in a host such as E. coli.

[0254] Next, a marker cassette is produced to be inserted between the 5' terminal side region and the 3' terminal side region on the vector. The marker cassette is manufactured so that a streptomycin sensitive gene functional in the hydrogenophilus bacterium and a positive selection marker gene functional in the hydrogenophilus bacterium are adjacent to each other, or a DNA of about 10,000 base pairs or less is inserted therebetween.

[0255] Next, if appropriate, the marker box is inserted between the 5' terminal side region and the 3' terminal side region of the mutated target gene on the vector into which the DNA fragment formed by the mutated target gene of the hydrogenophilic bacterium has been inserted. In addition, if appropriate, the host is transformed with the obtained vector according to a conventional method, and the vector is extracted from the transformant.

[0256] Next, where appropriate, the vector is linearized by cutting between the 5' terminal side region and the 3' terminal side region of the mutated target gene with a restriction enzyme, thereby providing a marker DNA fragment. The cutting is performed so that the marker box is in a state where it is inserted between the 5' terminal side region and the 3' terminal side region of the mutated target gene.

[0257] The gene of (7), (8) or (9) above can be used as a streptomycin-sensitive gene functional in bacteria of the genus Hydrogenophilus.

[0258] Until now, no positive selection marker gene that is functional in hydrogenophilic bacteria has been known. In the present invention, it has been found that the hygromycin resistance gene of (10), (11) or (12) below is functional in hydrogenophilic bacteria and can be used as a positive selection marker.

[0259] (10) a hygromycin resistance gene formed by the base sequence shown in SEQ ID NO: 4;

[0260] (11) a gene consisting of a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more, more preferably 99% or more identity with the base sequence shown in SEQ ID NO: 4, which encodes a polypeptide having an activity of providing hygromycin resistance to a bacterium of the genus Hydrogenophilus; or

[0261] (12) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 4, the gene encoding a polypeptide having an activity of imparting hygromycin resistance to a bacterium of the genus Hydrogenophilus.

[0262] The fact that the gene to be tested is a gene encoding a polypeptide having an activity of providing hygromycin resistance to a bacterium of the genus Hydrogenophilus is confirmed by the fact that the growth of the bacterium of the genus Hydrogenophilus into which the gene to be tested is inserted in the presence of hygromycin (100 μg / mL) is improved as compared with the wild-type bacterium of the genus Hydrogenophilus before the introduction of the gene to be tested (i.e., the fact that the wild-type bacterium of the genus Hydrogenophilus before the introduction of the gene to be tested is not able to grow in the presence of hygromycin (100 μg / mL)).

[0263] In the present invention, it has also been found that the kanamycin resistance gene of (13), (14) or (15) described below is functional in bacteria of the genus Hydrogenophilus and can be used as a positive selection marker.

[0264] (13) a kanamycin resistance gene formed by the base sequence shown in SEQ ID NO: 5 or 6;

[0265] (14) a gene consisting of a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more, more preferably 99% or more identity with the base sequence shown in SEQ ID NO: 5 or 6, which encodes a polypeptide having an activity of providing kanamycin resistance to a bacterium of the genus Hydrogenophilus; or

[0266] (15) A gene which hybridizes under stringent conditions with a DNA formed by a base sequence complementary to the base sequence shown in SEQ ID NO: 5 or 6, the gene encoding a polypeptide having an activity of imparting kanamycin resistance to a bacterium of the genus Hydrogenophilus.

[0267] The fact that the gene to be tested is a gene encoding a polypeptide having an activity of providing kanamycin resistance to a bacterium of the genus Hydrogenophilus is confirmed by the fact that the growth of the bacterium of the genus Hydrogenophilus into which the gene to be tested is inserted is improved in the presence of kanamycin (50 μg / mL) as compared with the wild-type bacterium of the genus Hydrogenophilus before the introduction of the gene to be tested (for example, the fact that the wild-type bacterium of the genus Hydrogenophilus before the introduction of the gene to be tested is not able to grow in the presence of kanamycin (50 μg / mL)).

[0268] ( B) Positive selection

[0269] In selecting each recombinant in which the target gene on the genome of the hydrogenophilus bacterium has been replaced with the marker DNA fragment by homologous recombination with the marker DNA fragment, it is appropriate to select each recombinant that has acquired the characteristics of the positive selection marker, for example, each recombinant that has become hygromycin-resistant or kanamycin-resistant.

[0270] In order to perform counter-selection using a streptomycin-sensitive gene as a counter-selection marker in the next step, a streptomycin-resistant strain of a hydrogenophilic bacterium is used as a host. When a bacterial suspension of a hydrogenophilic bacterium is applied to a solid medium containing about 50 μg / mL to about 500 μg / mL of streptomycin and cultured, streptomycin-resistant strains generally appear at a certain ratio.

[0271] The operation of introducing the marker DNA fragment into the streptomycin-resistant strain is carried out, and the characteristic of the positive selection marker is used as an indicator to select each recombinant in which the target gene on the genome of the streptomycin-resistant strain has been replaced by the marker DNA fragment. The marker DNA fragment to be introduced can also have additional nucleotides added to its two ends (5' end of the 5' end side region of the target gene of the mutation and 3' end of the 3' end side region of the target gene of the mutation). The recombinant to be obtained is streptomycin sensitive.

[0272] Introduction of DNA into hydrogenophilus bacterial cells can be performed by a known method, such as a calcium chloride method, a calcium phosphate method, a DEAE-dextran-mediated transfection method, or an electric pulse method.

[0273] ( C) Anti-selection

[0274] Next, in order to delete the marker cassette inserted into the mutated target gene, homologous recombination is performed using a DNA fragment formed from the mutated target gene.

[0275] By cutting in a way that the target gene of the mutation is not separated, the vector of the target gene of the mutation of the genus Hydrogenophilus is linearized. Where applicable, the linear DNA fragment is introduced into the streptomycin-sensitive strain (each recombinant in which the target gene has been replaced by the marker DNA fragment), and the strain that has become streptomycin-resistant is selected. It is also preferred to select a strain with streptomycin resistance and the characteristic of the positive selection marker. Therefore, a recombinant in which the marker box inserted into the target gene of the mutation has been eliminated is obtained, i.e., a recombinant in which a mutation of interest is introduced into the target gene.

[0276] Another example of a method for producing a recombinant hydrogenophilic bacterium using the above-mentioned counter-selection marker is a method comprising the following steps (D) to (G) (the second method, Figure 2 ):

[0277] (D) producing a labeled circular DNA comprising a DNA fragment of a mutated target gene of a bacterium of the genus Hydrogenophilus and a marker cassette, wherein a DNA fragment comprising a streptomycin-sensitive gene and a DNA fragment comprising a gene for a positive selection marker are linked to each other;

[0278] (E) performing an operation of introducing the marker circular DNA into a streptomycin-resistant strain of the hydrogenophilus bacterium, and then selecting each recombinant in which the marker circular DNA is linearized and inserted into the target gene of the streptomycin-resistant strain using the presence of the characteristic of the positive selection marker as an indicator;

[0279] (F) selecting each recombinant in which the region of the marker DNA inserted into the genome has been eliminated from the recombinants using streptomycin resistance as an indicator; and

[0280] (G) selecting a recombinant having a mutation introduced into the target gene from the recombinants selected in step (F).

[0281] ( D) Production of labeled circular DNA

[0282] A circular DNA containing a target gene of the genus Hydrogenophilus into which a mutation is introduced and the marker cassette is produced. The marker cassette is as described in step (A) of the first method. In suitable cases, the overall size of the circular DNA is about 15,000 base pairs or less.

[0283] ( E) Positive selection

[0284] In the case of being suitable, the operation of introducing the obtained marker circular DNA into the streptomycin-resistant strain of the hydrogenophilus bacterium is carried out, and each recombinant of the characteristic of the positive selection marker is selected, for example, each recombinant of hygromycin resistance or kanamycin resistance is changed. Therefore, the marker circular DNA is selected to be linearized and inserted into each recombinant in the target gene on the genome of the hydrogenophilus bacterium as a result of homologous recombination with the 5' end side region or the 3' end side region of the target gene. That is to say, on the genome of each of these recombinants, the DNA containing the marker box is inserted between the target gene of the mutation and the naturally occurring target gene. Therefore, the recombinant to be obtained is streptomycin sensitive.

[0285] ( F) Anti-selection

[0286] Each strain that has become streptomycin-resistant, preferably each strain that is streptomycin-resistant and has lost the characteristics of the positive selection marker, is selected from the obtained recombinants, and each recombinant in which the region between the mutated target gene and the naturally occurring target gene on the genome has been eliminated by a second homologous recombination between the two genes is obtained. The target gene on the genome of each of the recombinants to be obtained is a naturally occurring target gene in some cases and a mutated target gene in other cases. When the homologous recombination occurs in the same region as the region where the first homologous recombination occurs in the 5' terminal side region or the 3' terminal side region of the target gene, the obtained target gene is wild type; when the homologous recombination occurs in a region different from the region where the first homologous recombination occurs in the 5' terminal side region or the 3' terminal side region of the target gene, the obtained target gene has the mutation introduced therein.

[0287] ( G) Selection of recombinants with mutated target genes

[0288] From the recombinants obtained in step (F), a strain having a mutation of interest introduced into the target gene is selected. Where appropriate, the base sequence of the target gene of the obtained recombinant is determined to confirm the introduction of the mutation.

[0289] When the target gene is the acetolactate synthase III small subunit gene and the target gene of the mutation of interest is a gene encoding a mutated acetolactate synthase III small subunit having acetolactate synthase III small subunit activity and suppressing feedback inhibition by valine, it is appropriate to select a strain that has enhanced growth in the presence of valine compared to a wild-type strain of a hydrogenophilus bacterium that has undergone the mutation.

[0290] The mutant acetolactate synthase III small subunit gene formed by the base sequence in which GGC at bases 40 to 42 of SEQ ID NO: 2 is replaced by GAC contains a recognition sequence for restriction enzyme MluI as a result of the mutation. Therefore, for the recombinant obtained in step (F), it is appropriate to amplify the target gene by colony PCR and confirm that the amplified DNA fragment is cleaved by restriction enzyme MluI.

[0291] As described above, the combination of the positive selection marker and the counter selection marker, each of which is functional in a bacterium of the genus Hydrogenophilus, enables modification of genes on the genome by homologous recombination.

[0292] The recombinant hydrogenophilus bacterium having enhanced valine-producing ability of the present invention can also be produced by the above method.

[0293] The number of positive selection markers that can be used in hydrogenophilic bacteria is limited, but by combining the positive selection marker with a counter selection marker, recombination operations can be repeatedly performed any number of times, so that multiple genes on the genome can be modified.

[0294] Hydrogenophilus bacteria grow under autotrophic conditions. However, since they can also grow under heterotrophic conditions, the culture medium used to culture the host or hydrogenophilus bacteria recombinant can be an inorganic culture medium or an organic culture medium. An organic culture medium containing sugar, organic acid, amino acid, etc. can be used. The pH of the culture medium can be adjusted to about 6.2 to 8.

[0295] In any case, cultivation can be carried out while supplying a gas mixture containing hydrogen, oxygen and carbon dioxide, preferably a gas mixture consisting of hydrogen, oxygen and carbon dioxide. When using an organic culture medium, a gas mixture containing hydrogen, oxygen and carbon dioxide, such as air, can be used for ventilation. When carbon dioxide gas is not supplied, a culture medium containing carbonate as a carbon source can be used. The mixed gas can be trapped in or continuously supplied to an airtight culture container, and shaking culture can be utilized to dissolve the gas in the culture medium. Alternatively, the culture container can be airtight or open type, and the mixed gas can be dissolved in the culture medium by bubbling.

[0296] The volume ratio of hydrogen, oxygen and carbon dioxide (hydrogen: oxygen: carbon dioxide) in the supplied gas is preferably 1.75 to 7.5: 1: 0.25 to 3, more preferably 5 to 7.5: 1: 1 to 2, more preferably 6.25 to 7.5: 1: 1.5. Hydrogenophilus bacteria are thermophilic bacteria, so the culture temperature is preferably 35 to 55° C., more preferably 37 to 52° C., and even more preferably 50 to 52° C.

[0297] Example

[0298] ( 1) Acquisition of streptomycin-resistant strains

[0299] Thermoflavin Hydrogenophilus thermoymphalus TH-1 strain (NBRC 14978) (hereinafter sometimes referred to as "TH-1 strain") was inoculated using a platinum loop in a test tube in which 5 mL of A-liquid medium [3.0 g (NH4)2SO4, 1.0 g KH2PO4, 2.0 g K2HPO4, 0.25 g NaCl, 0.014 g FeSO4·7H2O, 0.5 g MgSO4·7H2O, 0.03 g CaCl2, 4.0 mg MoO3, 28 mg ZnSO4·7H2O, 2.0 mg CuSO4·5H2O, 4.0 mg H3BO3, 4.0 mg MnSO4·5H2O and 4.0 mg CoCl2·6H2O dissolved in 1 L of distilled water (pH 7.0) was placed. 7.0)], the test tube was filled with a mixed gas of H2:O2:CO2=7.5:1:1.5, and cultured with shaking at 50°C. After 24 hours, the culture solution was applied to A-solid medium [3.0 g (NH4)2SO4, 1.0 g KH2PO4, 2.0 g K2HPO4, 0.25 g NaCl, 0.014 g FeSO4·7H2O, 0.5 g MgSO4·7H2O, 0.03 g CaCl2, 4.0 mg MoO3, 28 mg ZnSO4·7H2O, 2.0 mg CuSO4·5H2O, 4.0 mg H3BO3, 4.0 mg MnSO4·5H2O, 4.0 mg CoCl2·6H2O and 15 g agar dissolved in 1 L of distilled water (pH 7.0)] containing 500 μg / mL streptomycin, and cultured at 50°C for 60 hours in an incubator filled with a mixed gas of H2:O2:CO2=7.5:1:1.5.

[0300] As a result, the formation of 3 colonies could be recognized on the A-solid medium containing 500 μg / mL streptomycin. These grown strains were streptomycin-resistant strains of the TH-1 strain, and one of the strains was named NOC269 strain.

[0301] ( 2) Construction of the marker box

[0302] ( 2-1) Preparation of counter-selection marker

[0303] Genomic DNA was extracted from a wild strain of TH-1 strain (streptomycin-sensitive strain) according to a conventional method. Using the extracted genomic DNA as a template, a DNA fragment containing the rpsL gene was amplified by a PCR method, and the rpsL gene is a streptomycin-sensitive gene encoding ribosomal protein S12. The following primers were used in the PCR. PCR was performed by a conventional method using a "DNA thermal cycler" manufactured by Life Technologies Inc. and using KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.

[0304] Primers used to amplify the wild-type rpsL gene of the TH-1 strain:

[0305] (a-1)5'-AT ACGCGT CCTCCGATGCGTCGTAAGGGAAACGTC-3' (SEQ ID NO: 7)

[0306] (b-1)5'-ATA GTCGAC TTATTTCTTGCCCGCAGGCGGCGCCCG-3' (SEQ ID NO: 8)

[0307] Primer (a-1) has an MluI restriction enzyme site added thereto, and primer (b-1) has a SalI restriction enzyme site added thereto.

[0308] ( 2-2) Preparation of positive selection marker

[0309] Using the DNA of plasmid pJR225 (GenBank: K01193) [Gene, 25, 179-188 (1983)] containing the sequence of hygromycin resistance gene (hereinafter sometimes referred to as "hph") ​​as a template, a DNA fragment containing the hph gene was amplified by the PCR method. The following primers were used in the PCR. The PCR was performed by a conventional method using a "DNA thermal cycler" manufactured by Life Technologies Inc. and using KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.

[0310] Primers used to amplify the hph gene:

[0311] (a-2)5'-ATA CTCGAG GAGATGACGTTGGAGGGGCAAGGTCG-3' (SEQ ID NO: 9)

[0312] (b-2)5'-AT ACGCGT CTATTCCTTTGCCCTCGGACGAGTGCT-3' (SEQ ID NO: 10)

[0313] Primer (a-2) has an XhoI restriction enzyme site added, and primer (b-2) has an MluI restriction enzyme site added.

[0314] The reaction solution produced by each of the above PCR was subjected to electrophoresis using 1% agarose gel. As a result, a DNA fragment of about 0.5 kb corresponding to the rpsL gene was detected when the genomic DNA of the TH-1 strain was used as a template, and a DNA fragment of about 1.0 kb corresponding to the hph gene was detected when the pJR225 plasmid DNA was used as a template.

[0315] The thus prepared DNA fragment containing the rpsL gene and the DNA fragment containing the hph gene were cut with restriction enzyme SalI and restriction enzyme XhoI, respectively, and mixed with Escherichia coli plasmid vector pUC19 (GenBank: M77789.2) which had been cut with restriction enzyme SmaI, and then ligated to each other using T4 DNA ligase (manufactured by Takara Bio Inc.).

[0316] The obtained ligation solution was used to transform Escherichia coli JM109 by the calcium chloride method, and the transformant was applied to the LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL hygromycin. The bacterial strain grown on the medium was liquid cultured by a conventional method, plasmid DNA was extracted from the culture fluid, and the plasmid was cut with restriction enzyme MluI. The inserted fragment was thus identified. As a result, in addition to the DNA fragment of the pUC19 vector of about 2.7 kbp, a DNA fragment of about 1.5 kbp corresponding to the sequence of the rpsL gene and the hph gene connected to each other was found.

[0317] The constructed plasmid containing the marker cassette in which the rpsL gene and the hph gene were linked to each other was named pUC-Sm s ·Hm r .

[0318] ( 3) Construction of the mutant ilvH gene [ilvH(G14D)]

[0319] Using the genomic DNA of the wild strain of the TH-1 strain extracted in (2-1) as a template, DNA fragments of the 5'-side region and the 3'-side region containing the mutant ilvH gene [ilvH (G14D)] were each amplified by the PCR method. The following primers were used for the PCR. The primers contain a sequence corresponding to the G14D mutation, so when these primers are used to amplify the region of the ilvH gene, the DNA region of the mutant ilvH gene [ilvH (G14D)] into which the G14D mutation is introduced can be amplified. PCR was performed by a conventional method using a "DNA Thermal Cycler" manufactured by Life Technologies Inc. and using KODFX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.

[0320] Primers used to amplify the 5'-flanking region of the ilvH(G14D) gene:

[0321] (a-3)5'-AT GAATTC CCCGGAAGCGACAAGCAGTTCCTGGGG-3' (SEQ ID NO: 11)

[0322] (b-3)5'-ACA ACGCGT CCGATTCGTTTTCCACCAGCAGTGCA-3' (SEQ ID NO: 12)

[0323] Primer (a-3) has an EcoRI restriction enzyme site added, and primer (b-3) has an MluI restriction enzyme site added. The ninth nucleotide from the 5' end of SEQ ID NO: 12 in (b-3) is a mutation site.

[0324] Primers used to amplify the 3'-flanking region of the ilvH(G14D) gene:

[0325] (a-4)5'-CGG ACGCGT TGTCGCGCATCGCCGGGCTCTTTTCG-3' (SEQ ID NO: 13)

[0326] (b-4)5'-TT CTGCAG GCGCTTTGCCGCTTTGGTCCTGATGCA-3' (SEQ ID NO: 14)

[0327] Primer (a-4) has an MluI restriction enzyme site added, and primer (b-4) has a PstI restriction enzyme site added. The fourth nucleotide from the 5' end of SEQ ID NO: 13 in (a-4) is a mutation site.

[0328] The reaction solution generated by each of the above PCRs was subjected to electrophoresis using 1% agarose gel. As a result, about 1.0-kb DNA fragments corresponding to the 5'-side region and the 3'-side region of the ilvH (G14D) gene were detected, respectively.

[0329] The DNA fragment of the 5'-side region of the ilvH (G14D) gene thus prepared was cleaved with restriction enzymes EcoRI and MluI, and the DNA fragment of the 3'-side region of the mutant ilvH (G14D) gene thus prepared was cleaved with restriction enzymes MluI and PstI. The cleavage products were mixed with the pUC19 vector cleaved with restriction enzymes EcoRI and PstI, and then ligated to each other using T4 DNA ligase (manufactured by Takara Bio Inc.).

[0330] Escherichia coli JM109 was transformed with the obtained ligation solution by the calcium chloride method, and the transformant was applied to an LB agar medium containing 50 μg / mL ampicillin. The strain grown on the medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture fluid, and the plasmid was cut with restriction enzymes EcoRI and PstI. The inserted fragment was thus identified. As a result, in addition to the DNA fragment of the pUC19 vector of about 2.7 kbp, a DNA fragment of about 2.0 kbp corresponding to the sequence of the 5'-side region and the 3'-side region of the ilvH (G14D) gene connected to each other was found.

[0331] In this plasmid, the 5'-side region and the 3'-side region of the ilvH(G14D) gene are linked to each other, and ilvH(G14D) is cloned as a complete gene. The constructed plasmid containing the ilvH(G14D) gene is named pUC-ilvH(G14D).

[0332] ( 4) Construction of plasmid for labeling ilvH

[0333] The pUC-Sm prepared in (2) above was s ·Hm r Cut with restriction enzyme MluI and perform electrophoresis using 1% agarose gel. Then, an approximately 1.5-kbp DNA fragment of the marker box in which the rpsL gene and the hph gene are linked to each other is cut out from the agarose gel, and the gel is freeze-thawed. Thus, the DNA is recovered from the gel.

[0334] The recovered DNA fragment of the marker cassette was mixed with pUC-ilvH(G14D) produced in (3) which had been cleaved with restriction enzyme MluI, and then ligated to each other using T4 DNA ligase (manufactured by Takara Bio Inc.).

[0335] By calcium chloride method, transform Escherichia coli JM109 with the connection solution obtained, and transformant is applied to LB agar medium containing 50 μ g / mL ampicillin and 50 μ g / mL hygromycin.By ordinary method, the bacterial strain grown on the substratum is carried out liquid culture, plasmid DNA is extracted from the culture fluid, and the plasmid is cut with restriction enzyme MluI.Identify the fragment of the insertion thus.As a result, except the DNA fragmentation of pUC-ilvH (G14D) plasmid of about 4.7-kbp, the DNA fragmentation of about 1.5-kbp corresponding to the sequence of the marker box is also found.

[0336] The constructed plasmid for labeling ilvH was named pUC-ilvH(G14D)-Sm s ·Hm r .

[0337] ( 5) Introduction of the G14D mutation into the ilvH gene of the TH-1 strain

[0338] ( 5-1) Marking the ilvH gene with a marker cassette (positive selection)

[0339] The circular plasmid pUC-ilvH(G14D)-Sm prepared in (4) was cleaved with restriction enzymes EcoRI and PstI. s ·Hm r Linearization. The linear pUC-ilvH(G14D)-Sm s ·Hm r The NOC269 strain, which is a streptomycin-resistant strain of the TH-1 strain obtained in (1), was transformed by an electric pulse method (electroporation method). The transformant was applied to A-solid medium containing 100 μg / mL hygromycin and cultured at 50° C. for 60 hours in an incubator filled with a mixed gas of H2:O2:CO2=7.5:1:1.5.

[0340] Each strain grown on the A-solid medium was restreaked on A-solid medium containing 100 μg / mL hygromycin or 500 μg / mL streptomycin, and cultured at 50° C. for 60 hours in an incubator filled with a mixed gas of H 2 :O 2 :CO 2 = 7.5:1:1.5.

[0341] Using the hygromycin-resistant and streptomycin-sensitive strains obtained as a result of the aforementioned operation as a template, a DNA region containing the ilvH gene was amplified by a colony PCR method. The PCR used the following primers. The primers were a combination of primers (a-3) and (b-4) used in (3), and using the genomic DNA of the wild-type TH-1 strain as a template, an approximately 2.0-kbp DNA region containing the ilvH gene was amplified by PCR. The PCR was performed by a conventional method using a "DNA Thermal Cycler" manufactured by Life Technologies Inc. and using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as a reaction reagent.

[0342] Primers used to amplify the region containing the ilvH gene:

[0343] (a-3)5'-AT GAATTC CCCGGAAGCGACAAGCAGTTCCTGGGG-3' (SEQ ID NO: 11)

[0344] (b-4)5'-TT CTGCAG GCGCTTTGCCGCTTTGGTCCTGATGCA-3' (SEQ ID NO: 14)

[0345] The resulting reaction solution was subjected to electrophoresis using 1% agarose gel. As a result, an approximately 3.5-kbp DNA fragment corresponding to a sequence in which a marker cassette was inserted into ilvH was detected. The strain in which the marker cassette was inserted into the ilvH gene, i.e., the ilvH gene was labeled with the marker, was named NOC278 strain.

[0346] ( 5-2) Replacement of the ilvH gene with the ilvH(G14D) gene (counter-selection)

[0347] The circular plasmid pUC-ilvH (G14D) prepared in (3) was linearized by cutting with restriction enzymes EcoRI and PstI. The obtained linear pUC-ilvH (G14D) was used to transform the NOC278 strain, which is a strain in which the ilvH gene obtained in (5-1) was marked, by an electric pulse method (electroporation method). The transformant was applied to A-solid medium containing 500 μg / mL streptomycin and cultured at 50° C. for 60 hours in an incubator filled with a mixed gas of H2:O2:CO2=7.5:1:1.5.

[0348] Each strain grown on the A-solid medium was restreaked on A-solid medium containing 100 μg / mL hygromycin or 500 μg / mL streptomycin, and cultured at 50° C. for 60 hours in an incubator filled with a mixed gas of H 2 :O 2 :CO 2 = 7.5:1:1.5.

[0349] Using the hygromycin-sensitive and streptomycin-resistant strains obtained as a result of the aforementioned operation as templates, the DNA region containing the ilvH gene was amplified by the colony PCR method. The colony PCR was performed by the same method as in (5-1).

[0350] The resulting reaction solution was subjected to electrophoresis using 1% agarose gel. As a result, the same approximately 2.0-kbp DNA fragment as in the case of PCR using the genomic DNA of the wild-type TH-1 strain as a template was detected. That is, this corresponds to the sequence from which the marker box has been removed.

[0351] The amplified about 2.0-kbp DNA fragment was cut with restriction enzyme MluI and subjected to electrophoresis using 1% agarose gel, and as a result, two about 1.0-kbp DNA fragments that would be produced in the case of cutting at the MluI site introduced together with the G14D mutation in (3) were detected. The about 2.0-kbp DNA fragment that would be amplified in the case of using the genomic DNA of the wild-type TH-1 strain as a template does not contain any MluI site, and therefore is not cut even when reacting with the restriction enzyme MluI, and maintains a size of 2.0-kbp. Therefore, the strain in which two about 1.0-kbp DNA fragments were detected by cutting with MluI is a strain in which the ilvH gene has been replaced by the ilvH (G14D) gene, that is, the G14D mutation has been introduced into the ilvH gene. The strain having the G14D mutation in the ilvH gene was named NOC279 strain.

[0352] ( 6) Confirmation of desensitization of valine feedback inhibition

[0353] The NOC279 strain, a strain of the genus Hydrogenophilus having a G14D mutation in the wild-type ilvH gene, was inoculated into each test tube containing 5 mL of A-liquid culture medium containing 1 mM or 10 mM valine using a platinum loop, and the test tubes were filled with a mixed gas of H2:O2:CO2 = 7.5:1:1.5 and cultured with shaking at 50°C.

[0354] The wild-type Hydrogenophilus bacteria were inhibited from growing in the presence of 1 mM valine, but the NOC279 strain into which the G14D mutation was introduced into the gene encoding acetolactate synthase III was not inhibited from growing even in the presence of 10 mM valine. Therefore, the NOC279 strain was desensitized to feedback inhibition by valine.

[0355] ( 7) Production of valine

[0356] The NOC279 strain, a strain of Thermoflavin Hydrogenophilus having a G14D mutation in the ilvH gene, was inoculated into A-liquid medium using a platinum loop and cultured with shaking at 50° C. for 30 hours while supplying a mixed gas of H 2 : O 2 : CO 2 = 7.5:1:1.5.

[0357] After the culture, valine in the culture supernatant obtained by centrifugation (4°C, 15,000 rpm, 1 minute) was quantified, and as a result, 9 mM valine was found to be produced in the culture supernatant. When the wild-type thermoflavour hydrogenophilus TH-1 strain was similarly cultured and valine in the culture supernatant was quantified, the result was 0.1 mM or less.

[0358] ( 8) Preserved strains

[0359] The thermoflavour hydrogenophile NOC279 strain is deposited in the National Institute of Technology and Evaluation (NITE Patent Microorganism Collection, Kazusakamatari 2-5-8, Kisarazu, Chiba Prefecture, Japan (Postal Code 292-0818)). For the thermoflavour hydrogenophile NOC279 strain, the accession number is BP-02829, and the receiving date is November 22, 2018. Therefore, the strain is available for public use.

[0360] In addition, all strains described in this specification are internationally deposited under the Budapest Convention, or are owned by an organization that provides the strains without any terms or conditions, or are sold, or can be obtained by anyone by following certain procedures, and therefore, these strains are available to the general public.

[0361] Industrial Applicability

[0362] The recombinant of the present invention effectively produces valine using carbon dioxide as the sole carbon source, and therefore, it can efficiently produce valine that can be used for medicines, foods, cosmetics, animal feed additives, etc., while solving global warming caused by increased carbon dioxide emissions. Sequence Listing <110> Utilization of Carbon Dioxide Institute Co., Ltd. <120> Genetic recombinant of hydrogenophilic bacteria with enhanced valine production ability <130> FRP0014WO <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 163 <212> PRT <213> Hydrogenophilus thermoluteolus <400> 1 Met Arg His Val Ile Ala Leu Leu Val Glu Asn Glu Ser Gly Ala Leu 1 5 10 15 Ser Arg Ile Ala Gly Leu Phe Ser Ala Arg Gly Tyr Asn Ile Glu Ser 20 25 30 Leu Thr Val Ala Pro Thr Glu Asp Glu Ser Leu Ser Arg Met Thr Ile 35 40 45 Val Thr Thr Gly Ser Asp Asp Val Ile Glu Gln Ile Thr Lys Gln Leu 50 55 60 Asn Lys Leu Ile Asp Thr Val Lys Val Val Asp Leu Ser Glu Ser Ala 65 70 75 80 His Ile Glu Arg Glu Leu Met Met Val Lys Val Arg Ala Val Gly Ser 85 90 95 Asp Arg Glu Glu Leu Lys Arg Leu Ala Asp Ile Phe Arg Ala Arg Ile 100 105 110 Its Asp Is Thr Asp Thr Thr Tyr Is Ile Glu Leu Thr Gly Asn Gln 115 120 125 Gln Lys Leu Asn Ala Phe Leu Ala Ala Ile Ser Pro Ser Leu Ile Leu 130 135 140 Glu Thr Val Arg Ser Gly Val Cys Gly Ile Ala Arg Gly Glu Arg Met 145 150 155 160 Leu Lys Ala <210> 2 <211> 492 <212> DNA <213> Hydrogenophilus thermoluteolus (Hydrogenophilus thermoluteolus) <400> 2 atgagacacg tgattgcact gctggtggaa aacgaatcgg gcgcgttgtc gcgcatcgcc 60 gggctctttt cggcgcgtgg ctacacatc gatcgctca ccgtcgcacc gaccgaagac 120 gagagcctgt cgcggatgac gatcgtcacc accggttcgg acgacgtgat cgaacagatc 180 accaagcagc tcaaac gatcgaacg gtcaagtgg tcgaccctc cgaatccgcg 240 cacatcgagc gcgaactgat gatggtgaag gtccgtgccg tgggatcaga ccgcgaagg 300 ctcaaacgcc tggccgacat cttccgcgcc cggatcatcg acgtcaccga caccacctac 360 gtgatcgaac tcaccggcaa ccagcagaag ctcaacgcgt ttctggcggc gatctcgccc 420 agtctcatct tggagacggt gcgtagcggc gtctgcggca ttgcccgtgg cgagcgtatg 480 ttgaaagcct ga 492 <210> 3 <211> 396 <212> DNA <213> Hydrogenophilus thermoluteolus <400> 3 atgccaacca tcaaccagtt ggtgcgtcgt ccgcggaaaa cggcgccga aaagagcaaaa 60 gtgccggcgt tgcagggatg tccgcaaaaa cgaggcgtgt gtacgcgcgt ctataccacg 120 acgccgaaaa agccgaactc ggcccttcgt aaggtcgcga aagtgcgttt gaccaacggt 180 tacgaggtga tttcgtacat cggcggcgaa gggcacaatc tgcaagaaca ctcggtggtg 240 ctgattcgtg gcggccgggt gaaagacctg ccgggtgtgc gttaccacat cgtgcgcggt 300 tcgctcgact tgcaaggggt caaggaccgt aagcaagggc gttccaagta cggggcgaag 360 cgtccgaagc cgggcgccgc tgcgggcaag aaataa 396 <210> 4 <211> 1026 <212> DNA <213> Escherichia coli <400> 4 atgaaaaagc ctgaactcac cgcgacatct gtcgagaagt ttctgatcga aaagttcgac 60 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat 360 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg 780 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020 gaatag 1026 <210> 5 <211> 795 <212> DNA <213> Escherichia coli <400> 5 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactc 180 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg gatgcccgac 540 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780 gacgagttct tctga 795 <210> 6 <211> 762 <212> DNA <213> Staphylococcus aureus <400> 6 atgaatggac caataataat gactagagaa gaaagaatga agattgttca tgaaattaag 60 gaacgaatat tggataata tggggatgat gttaaggcta ttggtgttta tggctctctt 120 ggtcgtcaga ctgatgggcc ctattcggat attgagatga tgtgtgtcat gtcaacagag 180 gaagcagagt tcagccatga atggacaacc ggtgagtgga aggtggaagt gaattttgat 240 agcgaagaga ttctactaga ttatgcatct caggtggaat cagattggcc gcttacacat 300 ggtcaatttt tctctatttt gccgatttat gattcaggtg gatacttaga gaaagtgtat 360 caaactgcta aatcggtaga agcccaaacg ttccacgatg cgatttgtgc ccttatcgta 420 gaagagctgt ttgaatatgc aggcaaatgg cgtaatattc gtgtgcaagg accgacaaca 480 tttctaccat ccttgactgt acaggtagca atggcaggtg ccatgttgat tggtctgcat 540 catcgcatct gttatacgac gagcgcttcg gtcttaactg aagcagttaa gcaatcagat 600 cttccttcag gttatgacca tctgtgccag ttcgtaatgt ctggtcaact ttccgactct 660 gagaacttc tggaatcgct agagaatttc tggaatggga ttcaggagtg gacagaacga 720 cacggatata tagtggatgt gtcaaaacgc ataccatttt ga 762 <210> 7 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 7 atacgcgtcc tccgatgcgt cgtaagggaa acgtc 35 <210> 8 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 8 atagtcgact tatttcttgc ccgcagcggc gcccg 35 <210> 9 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 9 atactcgagg agatgacgtt ggaggggcaa ggtcg 35 <210> 10 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 10 atacgcgtct attcctttgc cctcggacga gtgct 35 <210> 11 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 11 atgaattccc cggaagcgac aagcagttcc tgggg 35 <210> 12 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 12 acaacgcgtc cgattcgttt tccaccagca gtgca 35 <210> 13 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 13 cggacgcgtt gtcgcgcatc gccgggctct tttcg 35 <210> 14 <211> 35 <212> DNA <213> Artificial sequence <220> <223> PCR primers <400> 14 ttctgcaggc gctttgccgc tttggtcctg atgca 35

Claims

1. A mutant acetolactate synthase III small subunit, which is formed by a mutant amino acid sequence in which Gly at the 14th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is replaced by Asp.

2. A mutant acetolactate synthase III small subunit gene, which is formed by a mutant base sequence in which GGC at bases 40 to 42 in the base sequence shown in SEQ ID NO: 2 is replaced by GAT or GAC.

3. A bacterium of the genus Hydrogenophilus, which produces the mutant acetolactate synthase III small subunit according to claim 1, wherein the bacterium is obtained by replacing the acetolactate synthase III small subunit gene on the genome with a gene encoding the mutant acetolactate synthase III small subunit according to claim 1.

4. A hydrogenophilus bacterium comprising the mutant acetolactate synthase III small subunit gene according to claim 2, wherein the bacterium is obtained by replacing the acetolactate synthase III small subunit gene on the genome with the mutant acetolactate synthase III small subunit gene according to claim 2.

5. A method for producing valine, comprising the step of culturing the hydrogenophilic bacterium according to claim 3 or 4 using carbon dioxide as a sole carbon source.

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