WO2007013695A1 - Method of imparting glycerol-assimilation ability to bacterium - Google Patents

Abstract

It is intended to provide a means of imparting an ability to assimilate glycerol to a microorganism. Namely, a method which comprises transferring a glycerol intake protein gene into a bacterium to thereby impart an ability to assimilate glycerol to the bacterium.

Description

 Method of imparting glycerin assimilation ability to bacteria Technical Field

 The present invention relates to a method for imparting glycerin assimilation ability to a bacterium, a recombinant bacterium having a glycerin assimilation ability, and a fermented product or a reaction product using the recombinant bacterium. On the method.

 Light

 Background art

 In the surfactant industry and the biodiesel fuel industry, glycerin is discharged in large quantities as a by-product. Normally glycerin is used in the manufacture of nitroglycerin and other pharmaceuticals and cosmetics, but the glycerin discharged as a by-product is in the form of an aqueous solution containing impurities, so it can be used as it is for the above use! / The current situation is that it cannot be discarded. If glycerin, which is a waste material, can be used effectively in this way, it is highly desirable from the viewpoint of effective use of resources such as global environmental problems.

 On the other hand, to date, most of the carbohydrates derived from agricultural products are used as fermentation raw materials in the production of useful products by microorganisms. Coryneform bacteria widely used for fermentative production of useful substances such as amino acids and organic acids also use glucose as a carbon source. However, since the price of sugar derived from agricultural products is on the rise in the future, alternative fermentation raw materials are required. As a method for imparting to microorganisms the ability to assimilate carbon sources other than sugar, a methanol dehydrogenase gene is introduced into coryneform bacteria, and the hexulose phosphate synthase gene and the phosphohexoisomerase gene are introduced. There has been reported a method for imparting methanol-assimilating ability to the same bacterium by introducing the bacterium (Japanese Patent Application Laid-Open No. 2000-0 2 6 1 1 50). However, it is not known to impart glycerin assimilation ability to microorganisms.

The glycerin aqueous solution containing impurities discharged as a by-product in the surfactant industry and the piodiesel fuel industry has no problem when used as a fermentation raw material. Thus, the production of useful substances using glycerin as a raw material is desired not only from the viewpoint of developing fermentation raw materials other than sugar, but also from the viewpoint of effective use of waste. Disclosure of the invention

 An object of the present invention is to provide means for imparting glycerol utilization ability to microorganisms. As a result of intensive studies to solve the above problems, the present inventors have found that the bacterium acquires glycerin assimilation ability by introducing a gene encoding a glycerin uptake protein into the bacterium. The invention has been completed. '

 That is, the present invention includes the following inventions.

 (1) A method for imparting darinserin utilization ability to a bacterium by introducing a glycerin uptake protein gene into the bacterium.

 (2) The method according to (1), wherein the bacterium is a coryneform bacterium.

 (3) The method according to (2), wherein the coryneform bacterium is Corynebacterium dartamicum.

(4) The method according to (1), wherein the bacterium is a bacterium belonging to the genus Ralstonia.

 (5) The method according to (4), wherein the 'Ralstonia bacterium is Ralstonia utropha. (6) The method described in '(1), wherein the bacterium is an Al force Ligenes bacterium.

 (7) The method according to (6), wherein the alkali genus bacterium is Alcaligenes faecalis. (8) The method according to any one of (1) to (7), wherein the glycerin uptake protein gene is an Escherichia coli-derived g 1 p F gene.

 (9) The method according to any one of (1) to (7), wherein the glycerin uptake protein gene is a g1pF gene derived from Corynebacterium diphtheria '.

 (10) The method according to any one of (1) to (9), wherein a glycerol kinase gene is further introduced into a bacterium.

 (11) A recombinant bacterium having a glycerol-assimilating ability, into which a gene encoding a glycerin uptake protein has been introduced.

 (12) The recombinant bacterium according to (11), which is a coryneform bacterium.

 (13) The recombinant bacterium according to (12), which is Corynebacterium dartamicum.

 (14) The recombinant bacterium according to (1 1), which is a Ralstonia bacterium.

 (15) The recombinant bacterium according to (14), which is Ralstonia 'uto' lofa.

 (16) The recombinant bacterium according to (1 1), which is an alkaligenus bacterium.

 (17) The recombinant bacterium according to (16), which is Al force Ligenes-Fae force squirrel.

(18) The recombinant bacterium according to any one of (1 1) to (17), wherein the glycerin uptake protein gene is an E. coli-derived g 1 p F gene. (19) The recombinant bacterium according to any one of (11) to (17), wherein the glycerin uptake protein gene is a corynepacterium diphtheria-derived gl.pF gene.

 (20) The recombinant bacterium according to any one of (11) to (19), further introduced with a glycerol kinase gene.

 (21) Fermentation production comprising culturing the recombinant bacterium according to any one of (11) to (20) in a medium containing glycerin, and obtaining a fermentation product or a reaction product from the culture Product or reaction product production method.

 The present invention includes the contents described in the claims, specification and drawings of Japanese Patent Application No. 200-5-221282 which is the basis of the priority of the present application. Brief Description of Drawings

 FIG. 1 shows the result of extracting a plasmid from an E. coli transformant in Example 1 and confirming the insert.

 FIG. 2 shows the result of confirming the introduction of the hybrid plasmid into the corynebacterium dartami force in Example 2.

 FIG. 3 shows the results of PCR amplification of the g 1 p F K gene using KO D-gene polymerase in Example 3.

 Fig. 4 shows the result of excising and collecting the band of g I pFK gene obtained by PCR amplification and immobilizing it on pCR4TOPO.

 FIG. 5 shows the result of confirming the insert of g ip FK_pCG100-pHSG398 plasmid in Example 3.

 Fig. 6 shows that the plasmid g 1 p.FK—pCGl OO—pHSG398, in which the Escherichia coli g l.pFK gene was introduced into the pCG100—pHSG398 shuttle vector, was electroporated into Corynebacterium dartamicum ATCC 13032 strain. Show the result. Hereinafter, the present invention will be described in detail.

In the method of the present invention, as a bacterium for introducing a glycerin uptake protein gene, a bacterium that does not have glycerin assimilation ability is usually used. In addition, a bacterium having a glycerol kinase gene is preferably used. Preferably coryneform bacterium, Ralstonia Use bacteria belonging to the genus or Alcaligenes.

 : Coryneform bacteria are a group of microorganisms as defined in B argeys Manual of Det rm inative B acteriology, 8, 599 (1 974), aerobic, gram positive, non-acidic, spore-forming ability The bacterium belonging to the genus Corynebacterium, which has been previously classified as the genus Brevibacterium, has now been classified into the genus Corynebacterium. Incorporated bacteria (Int. J. Sys t. B acteriol., 41, 255 (1 98 1)), and Brevibakyuterium bacterium belonging to the genus Corynepacteria are also closely related. In particular, the following ¾ forces are exemplified: Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium lilium (Corynebacterium glutamicum), Corynebacterium raelassecola Brevibacterium ammoniagenes (Corynebacterium glutamicum), Brevibacterium divaricatum (Corynebacterium glutamicum), Brevibacterium linens Brevibacterium f lavum (Corynebacterium glutamicum), Brevibacterium lactof ermentum (Corynebacterium glutamicum), Brevibacterium saccharolyticum ^ Brevibacterium thiogenitalis, Brevibacterium helvolum. As a bacterium to which a gene is introduced in the method of the present invention, a bacterium belonging to the genus Corynebacterium is preferably used, and more preferably f. Corynebacterium glutamicum is used.

 A bacterium belonging to the genus Ralstonia can also be used as a bacterium for introducing a gene. Examples of the genus Ralstonia include Ralstonia eutropha, Ralstonia solanacearum, Ralstonia metal ldurans, Ralstonia pickettii, Ralstonia bxalatica., Ralstonia insidiosa, Ralstonia raannitolilytica ^ Ralstonia syzygii. As a bacterium for introducing a gene in the method of the present invention, preferably Ralstonia eutropha is used.

In addition, Alcaligenes bacteria can also be used as a cell for gene introduction. Alkagenes bacteria include, for example, Alcaligenes faecalis, Alcaligenes denitrif icans, Alcaligenes eutrophus, Alcaligenes xylosoxidans, Alcaligenes aestus, Alcaligenes aquamarinus _N Alcaligenes aquatilis ^ Alcaligenes cupidus, Alcaligenes defragrantus, caligen Alcaligenes paradoxus, Alcaligenes piechaudii, Alcaligenes ruhlandii, Alcaligenes venustus, etc. are mentioned. In the method of the present invention, the bacterium into which the gene is introduced is preferably Alcaligenes faecalis. In investigating the properties of glycerin as a raw material for host production, the present inventors have clarified that Corynepacterium 'Glutamicum, Ralstonia' Eutropha and Alkaline Genus' fecalis in a medium using glycerin as a carbon source. I found that it did not show good growth. Corynebacterium dartamicum, Ralstonia utropha and Arka ligenes faecalis have the ability to assimilate glycerin, despite the presence of the glycerol kinase gene, which phosphorylates glycerin and leads to the center of metabolism. It was amazing not to do it. In one embodiment, the present invention is based on the above findings.

Glycerin uptake protein is a hydrophobic protein, and is thought to act to promote uptake of glycerin into cells by forming a channel that connects the outside and inside by taking a state of being inserted into the cell membrane. . In this effort, the Dari 'serine uptake protein gene to be introduced into bacteria is not particularly limited as long as it is a gene encoding a protein having a function of promoting the uptake of glycerin into cells, but the glycerin assimilation ability is not limited. Derived from microorganisms, preferably having g 1 p F gene, for example, Salraone 丄 1 &, Yersinia genus,, Shigella, Erwiniaj¾ _s Haemophilus ^ Pasteureila ¾, Mannheimia genus, Xylella 厲, Vibrio genus, Photobacterium genus, Pseudomonas, Francisella, Chromobacterium, Burkholderia, Bacillus 厲, Staphylococcus, Listeria, Lactococcus, Streptococcus, Lactobacillus, Enterococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Streptomyces j¾ _N Leifs , Rhodopirellula, Boirelia, Leptospira, Archaeoglobus, Escherichia Those that come, specifically, Salmonella typhi, Yersinia pestis, Shigella flexneri, Erwinia carotovora Haemophilus influenzae Pasteurella multocida, Mannheimia succiniciproducens, Xylella fastidiosa, Vibrio par ahaemo 1 yti cus,

Photobacteriura profundura, Pseudomonas aeruginosa, Pseudomonas put i da, Pseudomonas syringae, Francisella tularensis, Chromobacterium violaceura, Burkholderia mallei Bacillus subtilis, Bacillus halodurans ^ Bacillus anthracis _N Bacillus anthracis _N Bacillus cereus , Listeria monocytogenes, Listeria innocua, Lactococcus lactis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Lactobacillus plantarum N Lactobacillus acidophilus, Enterococcus faecal is N Clostridium acetobutylicum, Clostridium perfringens ^ Thermoanaerobacter tengcongensis, Mycoplasma genitaliunu Mycoplasma pneumoniae, Mycoplasma gallisepticum, Corynebacteriura diphtheriae, Streptomyces coelicolor, Examples include those derived from Streptomyces avermitilis, Leifsonia xyli, Bifidobacterium longum, Rhodopirellula baltica, Borrelia burgdorferi ^ Leptospira interrogans _s Archaeoglobus iu gidus, EschericJia coli (especially Escherichia coli K-12), etc. Also, those derived from Corynebacteriura diphtheria are preferably used. In the present invention, the gene includes nucleic acids, that is, DNA and RNA.

 Microorganisms with glycerin assimilation ability such as E. coli corynebacterium 'Diphtheria' have a g 1 p FK gene that is thought to be involved in glycerin uptake. Glycerin uptake protein (also called facilitator), gl pK codes for glycerol kinase. In the present invention, the glycerin-incorporating protein gene to be introduced into the bacterium is preferably a g 1 pF gene, particularly a g 1 p F gene derived from E. coli and a g 1 p F gene derived from Corynebacterium diphtheria. -Specific examples of the glycerin uptake protein gene include a gene comprising the nucleotide sequence represented by SEQ ID NO: 1 or 7. The glycerin uptake protein gene of the present invention includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 7. Here, “functionally equivalent” means that the protein encoded by the gene of interest has the same biological function and biochemistry as the protein encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 7. It has a functional function.

 Methods known to those skilled in the art to prepare genes that encode proteins that are functionally equivalent to a protein include the hybridization technology (S a mb roo k ', J etal., Molecular Cloning 2 nde). d., 9. 47-9. 58, Old Spring Harbor Lab. press (1 989)).

As a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 7, Examples thereof include a gene that hybridizes with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence of No. 1 or 7 under stringent conditions. The protein encoded by the gene has the activity of glycerin uptake protein, that is, the activity of promoting the uptake of glycerin into cells.

 Stringent conditions are 3 if the probe is labeled with the D I G gene 'L a b e l i n g k i t (Boehringer Mannheim, catalog number 1 1 7503 3).

D I G E a s y Hy b solution at 2 ° C (Boehringer Mannheim's power tag number 1 60

3558) The condition of washing the membrane in 0.—5 XSSC solution (containing 0.1% [WZV] SDS) at 50 ° C (1 XS SC is 0.1 5M NaC) l, 0.0 1 5M sodium quenate).

 That is, in the full length of the nucleotide sequence shown in SEQ ID NO: 1 or 7, various artificial treatments such as site-directed mutagenesis, random mutation by mutagen treatment, nucleic acid fragment mutation by deletion of restriction enzyme, deletion, ligation, etc. Thus, even if the sequence is partially changed, these variant genes hybridize under stringent conditions with a gene consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1 or 7, and A gene encoding a protein having an activity of promoting the uptake of glycerin into cells is included in the glycerin uptake protein gene regardless of the difference from the base sequence shown in SEQ ID NO: 1 or 7. '' In the present invention, the darling uptake protein gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, to the nucleotide sequence of SEQ ID NO: 1 or 7. Preferably, a gene consisting of a base sequence having at least 99% identity is also included.

 Examples of the glycerin uptake protein gene include a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8. The gene includes a gene encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8. “Functionally equivalent” means that the target protein has a biological function or biochemical function equivalent to that of the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8.

As a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8, the amino acid sequence of SEQ ID NO: 2 or 8 is deleted, added, inserted or substituted in one or several amino acids. And a protein consisting of the prepared amino acid sequence. The protein has the activity of the above glycerin uptake protein, that is, glycerin into the cell. Has activity to promote uptake.

 One or several amino acid deletions, additions, insertions or substitutions in the amino acid sequence of SEQ ID NO: 2 or 8 are commonly used techniques such as site-directed mutagenesis (Z o 1 1 er et al., Nu cleic Ac ids R es. 1 0 6478-6 500, 1 98 2).

 Here, the side chains of amino acids that constitute protein components differ in hydrophobicity, charge, size, etc., but they are essentially in the three-dimensional structure of the entire protein (also referred to as a three-dimensional structure). Several relationships that are highly conserved in the sense that they have no effect are known from empirical and physicochemical measurements. For example, conservative substitutions between different amino acid residues include glycine (G 1 y) and proline (Pro), glycine and alanine (A la) or norin (V a 1), leucine (L eu) and isoleucine (I 1 e), glutamic acid (G 1 u) and glutamine (G in), aspartic acid (A sp) and asparagine (A sn), cysteine (Cy s) and threonine (Th r) ', Substitution between amino acids such as threonine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg) is known.

 Therefore, even if the mutant protein is composed of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 or 8, the mutation is If the mutation is highly conserved in the three-dimensional structure of the amino acid sequence shown in SEQ ID NO: 2 or 8, and the mutant protein has the activity of the glycerin uptake protein, these mutations The gene encoding the type protein is also included in the glycerin uptake protein gene. Here, the term “several” means usually 2 to 5, preferably 2 to 3.

 In the present invention, the glycerin incorporation protein gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the amino acid sequence of SEQ ID NO: 2 or 8. More preferably, a gene encoding a protein consisting of an amino acid sequence having at least 99% identity is also included.

 In one embodiment, a glycerol kinase gene is further introduced into the bacterium. Glycerol kinase is a protein having an activity of phosphorylating glycerin, and has an activity of catalyzing the following reaction in the metabolism of glycerin.

Glycerol + AT P → L—Glyce mouth loupe 3—Phosphate + AD P The glycerol kinase gene is not particularly limited as long as it is a gene encoding a protein having an activity to phosphorylate glycerin, but it is derived from a microorganism having glycerin assimilation ability, preferably has a g 1 p K gene. For example, Escherichia, Sa 丄 moneila, Yersinia, Shigella, Erwinia, Haemophilus 禹, Pasteure 丄 la, Mannheiraia 禹, Xylella ¾, Pseudomonas, Francisella 厲, Chromobacteriura, Burkholderia 禹, Bacillus ノ、, Staphylococcus 禹, Listeria, Lactococcus, Streptococcus, Lactobacillus, Enterococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Corynebacterium, Streptomycesj¾, Lensoriia Genus, Shewanella, Ralstonia, Bordetella, Geobacter, Desulfovib genus rio, Bdellovibrio, Sinorhizobium, Agrobaoterium, Brucella, Bradyrhizobium, Rhodopseudoraonas, Silicibactei; Genus, Nocardia, Gloeobacter 厲, Chlorobium, Aquifex, Haloarcula, Pyrococcus, Bacteria Ru ¹ include those of. Specifically 0 勺 to f, Salmonella typhi, Yersinia pestis, Shigella f lexneri Erwinia carotovora, Haemophilus influenzae, Pasteurella mu 丄 tocida, Mannheiraia succinic iproducens, Xylella fastidiosa, .Pseudomonas aeruginosa ^ Burkholderia mallei, Bacillus subtilis, Bacillus halodurans ^ Bacillus anthracis Bacillus cereus, Bacillus thuringiensis, Bacillus licheniformis, Bacillus clausi i, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Listeria monocytogenes, Listeria incytoc arum Lactobacillus acidophilus, Enterococcus faeca 丄 is, Clostridium acetobutylicum Clostridium perfringens ^ Thermoanaerobacter tengcongensis, Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma galli'septicum, Corynebacterium diphtheria e, Streptorayces coelico'lor, Streptomyces avermitilis, Leifsonia xyli, Bifidobacterium longum, Rhodopirellula baltica, Borrelia burgdorferi Leptospira interrogans Archaeoglobus fulgidus, Escherichia coli K-12 ella ed, Echerichia coli K-wan ella , Desulfovibrio vulgaris, Bdellovibrio bacteriovorus, Sinorhizobium meliloti, Agrobacteriura tumefaciens, Brucella suis, Bradyrhizobiura japonicum _Λ Rhodopseudoraonas palustris, Silicibacter pomeroyi, Corynebacterium glutamicura, Nocardia farcinica, Gloeobacter violaceus, Chlorobium tepidum, In the present invention, those derived from E. coli are preferred. Further, those derived from Corynebacterium diphtheria are also preferably used.

 As the glycerol kinase gene to be introduced into bacteria in the present invention, the g 1 p K gene in the above g 1 p F K gene, particularly the g 1 p K gene derived from Escherichia coli and the g 1 p K gene derived from Corynebacterium diphtheria are preferable. The glycerol kinase gene is preferably derived from the same bacterium as the glycerin uptake protein gene to be introduced. When introducing the Escherichia coli-derived g 1 p F gene as a glycerin uptake protein gene, the E. coli-derived g 1 p K gene present in the same operon is introduced into Escherichia coli, which has an excellent ability to assimilate glycerin. Replacement bacteria can be obtained. In addition, when the g1 p F gene derived from Corynebacterium diphtheria is introduced as a glycerin uptake protein gene, it is derived from Corynebacterium diphtheria present in the same operon in Corynebacterium diphtheria. By introducing the K gene, a recombinant bacterium excellent in glycerin assimilation ability can be obtained. In addition, the g 1 p F gene and the g 1 p K gene should be introduced as g 1 p FK genes contained in g 1 p (glycerol phosphate phosphate) in E. coli or corynebacterium diphtheria. Is more preferable.

 Specific examples of the glycerol kinase kinase gene include a gene consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 9. The glycerol kinase gene also includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 9. Here, “functionally equivalent” is the same as above.

 'A gene that is functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 9 includes a gene that is highly pre-hybridized under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 9 Is mentioned. The protein encoded by the gene has glycerol phosphorylation activity. The stringent conditions are as described above.

That is, the entire length of the nucleotide sequence set forth in SEQ ID NO: 3 or 9 is subjected to various artificial treatments. Even if the sequence is partially changed, these mutant genes are hybridized under stringent conditions with a gene consisting of a base sequence complementary to the base sequence of SEQ ID NO: 3 or 9. Any gene that codes for a protein having glycerin phosphorylation activity is included in the glycerol kinase gene regardless of the difference from the nucleotide sequence shown in SEQ ID NO: 3 or 9.

 In the present invention, the glycerol kinase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the nucleotide sequence of SEQ ID NO: 3 or 9. Furthermore, a gene consisting of a nucleotide sequence having at least 99% identity is preferably included.

 The glycerol kinase gene also includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 or 10. The gene includes a gene encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4 or 10. The protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4 or 10 is a deletion, addition, or insertion of Γ or several amino acids in the amino acid sequence of SEQ ID NO: 4 or 1.0. Alternatively, a protein having a substituted amino acid sequence can be mentioned. The protein has the above-mentioned glycerol kinase activity, that is, glycerin phosphorylation activity.

 Even if it is a mutant protein consisting of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 4 or 10, If the mutation is highly conserved in the three-dimensional structure of the amino acid sequence described in No. 4 or 10, and the mutant protein has the activity of the glycerol kinase, these mutant types The gene encoding the protein is also included in the glyceose mouth kinase gene. Here, the term “several” means usually 2 to 5, preferably 2 to 3. Also, in the present invention, the glycerol kinase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the amino acid sequence of SEQ ID NO: 4 or 10. Also included is a gene encoding a protein consisting of an amino acid sequence having a sex, more preferably at least 99% identity.

If the target bacterium to be imparted with glycerin-utilizing ability does not have a glycerin kinase gene, it is necessary to introduce a glycerol kinase gene. However, if the target bacterium has a glycerol kinase gene, the glycerol kinase gene is not necessarily introduced. -In an embodiment, a glycerol triphosphate dehydrogenase gene is further introduced into the bacterium. Glycerol triphosphate dehydrogenase (also referred to as glycerin triphosphate dehydrogenase) is a protein having an activity of dehydrating glycerol triphosphate (also referred to as glycerin triphosphate).

The glycerol triphosphate dehydrogenase gene is not particularly limited as long as it is a gene encoding a protein having an activity of dehydrating glycerol triphosphate, but it is derived from a microorganism having glycerin assimilation ability, preferably g Those having an I p A gene, for example, Escherichia coli fc, Salmonella ^, Yersinia, Shigella, Haemophilus, Xanthomonas, Vibrio 禹, Pseudomonas, Methylococcus, Neisseria 晨, Ralstonia, Burkholderia, Bordetella , Nitrosomonas 晨, Helicobacter genus, Geobacter genus, 'Agrobacterium genus, Rhizobium genus, Brucella genus, Bradyrhizobium genus, Rhodopseudomonas genus, Rhodobacter genus, Zymoraonas genus, (jluconobacter genus, Bacillus genus, Sta List Streptococctis, Lactobacillus, Enterococcus, Clostridium, Mycoplasma, Mycobacterium, CorynebacteriumS, Nocardia sp, Streptomyces sp, Propionibacteriura ^, Bif idobacterium genus Thermus genus Aquifex genus Thermotoga genus Methanobacterium genus includes those forces ^s of from Itoda bacteria Archaeoglobus sp. Specifically Ho, Escherichia coli K- 12 MG1655, Salmonella enterica serovar Typhi CT18, Salmonella typhi murium T2, Yersinia pest is C092, Shigella f lexneri 2457T, Shigella sonnei, Haemophilus influenzae (serotype d), Haemophilus ducreyi, estrisona p. , Xanthomonas oryzae KACC10331, Vibrio cholerae, Vibrio vulnificus CMCP6, Vibrio parahaeraolyticus, Vibrio f ischeri, Pseudomonas aeruginosa, Pseudomonas put i da, Pseudomonas -syringae pv. Tomato DC3000, Pseudomonse lure solanacearum _N Ralstonia eutropha ^ Burkholderia mallei, Burkholderia pseu domallei K96243, Burkholderia xenovorans ^ Burkholderia cenocepacia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Nitrosomonas europaea, Nitrosospira multiformis, Helicobacter pylori 26695, Helicobacter hepaticer sulfurreducens, Geobacter metallireducens, Agrobacterium tumefaciens C58 (UWash / Dupont), Rhizobiura etli, Brucella melitensis, Brucella abortus, Bradyrhizobium japonicum, Rhodopseudoraonas palustris CGA009, Nitrobacter winogradskyi, Nitrobacter haraburgensis, Rhodobacter sphaeroides, Zyraomonas mobilis, Gluconobacter oxydans, Bacillus subtilis N Bacillus halodurans ^ Bacillus anthracis Ames, Bacillus cereus ATCC 14579, Bacillus thuringiensis, Bacillus licheniformis ATCC 14580, Bacillus clausi i, Staphylococcus aureus N315, Staphylococcus epiderraidis ATCC 12228,, .- Staphylococcus haemolyticcus sa lactis, Streptococcus pyogenes SF370 (serotype Ml), Streptococcus neumoniae TIGR4, Streptococcus agalactiae 2603 (serotype V), Streptococcus mutans, Streptococcus thermophilus CNRZ1066, LactoDacillus plantarum jo, Lactobacillus hnsonii, Lactobacillus acidophilus, Lactobacillus sakei, Lactobacillus sa丄lvarius, Lactobacillus delbrueckn, Ehterococcus faecalis, Clostridium acetobutyliGUin> Clostridium perfringens, Clostridium tetani E88, Mycoplasma pulmonis ^ Mycoplasma penetrans N Mycoplasma gallisepticum, Mycoplasma mycoides, Mycoplasma mobile, Mycoplasma synoviae, Mycoplasma capricolura, Mycobacterium tuberculosis H37Rv, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium paratuberculosis ^ Corynebacterium glut ami cum (Kyowa Hakko), Corynebacteriura eff iciens, Corynebacteriura jeikeium, Nocardia farcinica, Corynebacterium diphtherial Streptomyces coelicolor, Streptorayces' avermiti丄is, Propionibacterium acnes, Bifidobacterium longum _N Examples include those derived from Therraus thermophilus, Aquifex aeolicus, Therraotoga raaritima, Methanobacteriura thermoautotrophicum, Archaeoglobus fulgidus, etc. Rinepaku Teriumu-diphtheria (Corynebacteriura diphtheriae) from what is preferred.

 As the glycerol triphosphate dehydrogenase gene to be introduced into the bacterium in the present invention, the g1 p A gene derived from Corynebacterium diphtheria is preferable. The glycerol triphosphate dehydrogenase gene is preferably derived from a bacterium belonging to the same genus as the glycerin uptake protein gene to be introduced.

When introducing the glycerol triphosphate dehydrogenase gene, By introducing it as a g 1 p AFK gene contained in the same operon (g 1 p) in diphtheria and the like, a recombinant bacterium excellent in glycerin assimilation ability can be obtained.

 A specific example of the glyce mouth triphosphate dehydrogenase gene is a gene consisting of the base sequence represented by SEQ ID NO: 11. The glycerol kinase gene also includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 11. Here, “functionally equivalent” is the same as above.

 A gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 11 includes a gene that hybridizes with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 11 under stringent conditions. Can be mentioned. The protein encoded by the gene has glycerin triphosphate dehydrogenase activity. The stringent conditions are as described above.

 That is, even though the entire length of the base sequence shown in SEQ ID NO: 1 1 is partially altered by various artificial treatments-'these mutant genes have the bases of SEQ ID NO 1 1 If it is a gene that hybridizes with a gene consisting of a base sequence complementary to the sequence under stringent conditions and encodes a protein having glycerin triphosphate dehydrogenase activity, the base sequence shown in SEQ ID NO: 11 Regardless of the difference, it is contained in the glycerin triphosphate dehydrogenase gene.

 In the present invention, the glycerin triphosphate dehydrogenase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: 11. More preferably, a gene comprising a base sequence having at least 99% identity is also included.

 The glycerin triphosphate dehydrogenase gene also includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 12. The gene includes a gene encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 12. As a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1, one or several amino acids are deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO: 1 And a protein consisting of the prepared amino acid sequence. The protein has glycerin triphosphate dehydrogenase activity.

Even a mutant protein consisting of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 2 If the difference is a highly conserved mutation in the three-dimensional structure of the amino acid sequence described in SEQ ID NO: 12, and the mutant protein has the activity of glycerin triphosphate dehydrogenase, Genes encoding these mutant proteins are also included in the glycerin triphosphate dehydrogenase gene. Here, the term “several” means usually 2 to 5, preferably 2 to 3.

 In the present invention, the glycerin triphosphate dehydrogenase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the amino acid sequence of SEQ ID NO: 12. More preferably, a gene encoding a protein consisting of an amino acid sequence having at least 99% identity is also included.

 If the target bacterium that is to be glycerinated does not have the glycerin triphosphate dehydrogenase gene, it is necessary to introduce the glycerin triphosphate dehydrogenase gene. However, if the target bacterium has a glycerin triphosphate dehydrogenase gene, the glycerin triphosphate dehydrogenase gene need not necessarily be introduced.

 In addition, preferably, all of the genes described above are derived from bacteria belonging to the same genus as the bacterium to be introduced. For example, when a gene is introduced into a bacterium belonging to the genus Corynebacterium, it is preferable to introduce a gene derived from a bacterium belonging to the genus Corynebacterium, preferably Corynebacterium diphtheria. Preferably, it is introduced as the g 1 p F gene derived from Corynebacterium diphtheria, particularly as the g 1 p A F K gene contained in the glycerol phosphate operon derived from Corynebacterium diphtheria. Corynebacterium diphtheria has glycerin assimilation ability but was difficult to use because it is a pathogen. However, by introducing a glycerin uptake protein gene derived from Corynebacterium diphtheria into another Corynebacterium genus 'bacteria', it is possible to obtain a bacterium having excellent glycerin utilization ability and having no pathogenicity.

 Introducing a gene into a bacterium involves linking the above gene or a part thereof to an appropriate vector and introducing the resulting recombinant vector into a host so that the target gene can be expressed, or It can be carried out by inserting the gene of interest or a part thereof at any position on the genome by homologous recombination. “Part” refers to a part of each gene capable of expressing the protein encoded by each gene when introduced into a host.

A method for obtaining a desired gene from a bacterial genome by cloning is well known in the field of molecular biology. For example, if the gene sequence is known, A more suitable genomic library can be created and screened using a probe complementary to the desired gene sequence. Once the sequence is isolated, the DNA is amplified using standard amplification techniques such as polymerase, chain reaction (PCR) (US Pat. No. 4,683,202) and is suitable for transformation. DNA can be obtained. The glycerin uptake protein gene or glycerol kinase gene can be hybridized using a known synthetic glycerin uptake protein gene or a synthetic DNA primer appropriately designed based on the base sequence of the glycerol kinase gene as a cage. It can also be obtained by the PCR method or PCR method.

 Methods such as preparation of genomic DNA libraries used for gene cloning, hybridization, PCR, preparation of plasmid DNA, DNA cleavage and ligation, and transformation are described in Sambrook, J., Fritsch, E F., Ma niatis, T., Ο ο 1 ecular Cloning, Old Spring Harbor Laboratory Press, 1. 2 1 (1 98 9)-.

 The vector to which the gene is linked is not particularly limited as long as it can be replicated in the host cell, and examples thereof include plasmids, phages and cosmids. Preferably, a shuttle vector that is movable between the bacterium from which the gene is introduced and the bacterium from which the gene is introduced is used. When a glycerin-incorporating protein gene derived from E. coli is introduced into a coryneform bacterium, a shuttle vector that can move between E. coli and the coryneform bacterium is preferable. When a glycerin uptake protein gene derived from E. coli is introduced into Ralstonia bacteria, a shuttle vector that can move between E. coli and Ralstonia bacteria is preferred. When introducing a glycerin-incorporating protein gene derived from Escherichia coli into an alkaline genus bacterium, a shuttle vector that can move between Escherichia coli and the genus Algigen genus is preferable.

Specifically, pAM 3 30 derived from prebibataterium lactofamentum 2256 (JP 58-6 7699, Ag ric c. Biol. Chem. Vol. 48, 290 1-2903 ( 1 984) and Nu c 1 ei cAc ids S ymp Ser. Vol. 1 6, 26 5 -267 (1985)), Corynebacterium glutamicum ATCC 1 3 058 strain pHMl 5 1 9 (A gric. B iol. C he m. vol. 48, 29 0 1-2903 (1 984)), p CRY 30 (Ap pl. En viro n. Mi crobiol. Vo l. 5 7, 759-764 (1 99 1)), p CG 100 (Trautwetter & B lanco, J ournalof General M icrobio logy, Vo l. 1 3 7, 2093 -2 1 0 1 (1 9 9 1)) ρ CG 4 derived from Corynebacterium glutamicum T 2 50 (Japanese Patent Laid-Open No. 5 7- 1 8 3 79 9, J. B acterio 1. Vo l. 1 59, 306-3 1 1 (1 984)), pAG l, pAG3, p AG 14, p AG 50 (JP-A-62-166890), p EK0, pEC 5, p EKE x 1 (G enevo 1. 102, 93—98 (1 9 9 1)), etc., or hybrid plasmids derived from them can be used (T rau twe tter & B lanco, J ourna 1 of General Microbiology, ol. I d, 20 93— 2 1 01 (1 99 1)).

 In the above vector, in order to ensure that the inserted gene is expressed, an appropriate expression promoter is connected upstream of the gene. The expression promoter to be used is not particularly limited as long as it is not repressed by glycerin, and may be appropriately selected by those skilled in the art depending on the host. For example, when the host is Escherichia coli, T7 promoter, lac promoter, trp promoter, λ-PL promoter, etc. can be used, and when the host is a Bacillus genus, a SPO promoter or the like can be used. When introducing the E. coli-derived g 1 p F gene, the promoter (P glp FK) contained in E. coli g 1 p (glycerol 'phosphate operon) is used from the viewpoint of efficient expression of glycerin uptake protein. I like it. When introducing the g 1 p F gene derived from Corynebacterium diphtheria, from the viewpoint of efficient expression of glycerin-incorporating protein, the promoter contained in Corynebacterium diphtheria g 1 p (glycerol phosphate operon) (P glp FK) is preferably used. Also preferred are promoters that are induced by glycerin.

 The method for introducing the above vector into the host is not particularly limited. For example, the electric pulse method (Y. K'u rusu, eta 1., Ag ri c. B io 1. C he m. 54: 443 -447 (19 90)) 方法 Method using calcium ion (Proc. Natl. Acad. Sci. USA, 6 9, 21 10 (1 9 72)), Protoplast method (Japanese Patent Laid-Open No. 6-2483942) ), Electroporation (Nuclei cAcids Res., 16, 6, 1 27 (1 98 '8))) and the like.

In the method of inserting a target gene at an arbitrary position on the genome by homologous recombination, the target gene is inserted into a sequence homologous to the sequence on the genome together with a promoter, and this DNA fragment is inserted into the cell by electroporation. It can be carried out by introducing and causing homologous recombination. When introducing into the genome, use a DNA fragment linking the target gene and drug resistance gene. Then, strains that have undergone homologous recombination can be selected easily. In addition, a gene linked to a drug-resistant gene and a gene that is lethal under specific conditions is inserted into the genome by homologous recombination as described above, and then the drug-resistant gene is lethal under specific conditions. It is also possible to introduce the target gene using homologous recombination in a form that replaces the gene.

 A method for selecting a recombinant bacterium into which a target gene has been introduced is not particularly limited, but a method that allows easy selection of only a recombinant bacterium into which a target gene has been introduced is preferred. The culture conditions for recombinant bacteria can be set based on the conditions normally used in the art depending on the type of bacteria used as the host. However, in this case, the culture medium for recombinant bacteria must be glycerin. It is desirable to contain it as the sole carbon source.

 The glycerin assimilation test of recombinant bacteria can be carried out by culturing these recombinant bacteria in a medium containing glycerin and following changes in the glycerin uptake rate and bacterial cell amount. Measurement of the glycerin uptake rate by the recombinant bacteria can be carried out by measuring the concentration of glycerin remaining in the medium using an HPLC analysis. The change in the amount of bacterial cells can be measured by measuring the change in optical density at 60 nm with a spectrophotometer.

 The recombinant bacterium of the present invention obtained as described above is cultured in a medium, a fermentation product or a reaction product is produced and accumulated in the medium or bacterial cell, and the product is collected from the medium or bacterial cell. Thus, a fermentation product or a reaction product can be produced using glycerin.

Examples of fermentation products or reaction products to which the method of the present invention can be applied include substances produced by metabolism of glycerin and substances produced by utilizing energy produced by metabolism of glycerin. It is done. Specifically, for example, amino acids such as glutamic acid, lysine, threonine, phenylalanine, tryptophan, etc .; organic acids such as pyruvic acid, lactic acid, acetic acid surrounding organic acids such as pyruvic acid, lactic acid, acetic acid, kenic acid, fumaric acid Organic acids around the TCA cycle, such as malic acid, succinic acid, itaconic acid, glyceric acid, organic acids around glycerin such as 3-hydroxypropionic acid; alcohols such as ethanol, propanol, 1 3-Propanediol; vitamins such as vitamin C; and polymer substances such as various enzymes. The production of amino acids using bacteria can be carried out by methods known in the art except that a medium containing glycerin as a carbon source is used (for example, amino acid fermentation, Hiroshi Aida et al., Academic Publishing Center). The culture medium, culture medium and culture conditions can be set appropriately according to the type of bacteria used, but a normal medium containing a nitrogen source, inorganic ions and other organic micronutrients as necessary. Can be used.

 Glycerin is used as the carbon source. Along with glycerin, sugars such as glucose, lactose, galactose, fructose or starch hydrolysate, alcohols such as sorbitol, or organic acids such as fumaric acid, succinic acid or succinic acid may be used in combination.

 Here, glycerin as carbon can be used in the form of an aqueous solution containing impurities that is discharged in large quantities as a by-product in the surfactant and biodiesel fuel industries. Therefore, the present invention is also advantageous from the viewpoint that the by-products that have been discarded can be used effectively.

 As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

 As inorganic ions, potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added. As organic micronutrients, it is desirable to contain required amounts of L-homoserine, vitamin B1, etc. or yeast extract as required.

 Culturing is performed under conditions suitable for the growth of the bacteria to be used. In the case of coryneform bacteria, it is usually better to carry out the reaction under aerobic conditions for 16 to 96 hours, the culture temperature is 20 ° C to 45 ° C, and the pH is 5 to 8.5 during the culture. To control. For the adjustment, it is possible to use an inorganic or organic acidic or alkaline substance, as well as ammonia gas. When a thermophilic bacterium is used as a host, the culture * temperature can be cultured at 42 ° C. to 60 ° C. Culture can be performed by aerobic cell growth followed by anaerobic conditions. This method is particularly effective when producing organic acids such as lactic acid and succinic acid. In the case of producing succinic acid, it is preferable to add carbon dioxide and carbonate ions to the medium because the yield increases.

The collection of the fermentation product or reaction product from the culture medium after completion of the culture does not require a special method in the present invention. That is, it can be carried out by combining conventionally known ion exchange resin method, precipitation method and other methods. When glycerin is used as a carbon source, it may be possible to simplify the purification of the target substance and the waste liquid treatment process, compared to the case where saccharides derived from agricultural products are used. ■ BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail by way of examples. However, the scope of the present invention is not limited to the examples.

 (Example 1) Construction of hybrid plasmid

 For the preparation of hybrid plasmids, use the plasmid ρ CG 100 (3 kbp) derived from Corynepacterum 'Dartamicam ATCC 1 3058 strain and the plasmid pHSG398 (Takara Bio Inc.) derived from Escherichia coli. It was.

 PCG100 plasmid derived from Corynebacterium dartamicum ATCC 13058 strain was obtained and ligated with pHSG398 to prepare a hybrid plasmid.

1— 1. Obtaining PCG 100 plasmid

Cell pre-culture:

 Corynebacterium 'glutamicum ATCC 13058 strain was cultured with shaking in JCM26 medium (5 m' 1) at 30 ° C. The cultured Corynebacterium 'Dartamicam A TCC 13058 strain was seeded on a JCM26 plate and cultured at 30 ° C with shaking. JC M26 plate was inoculated with 1 loop in a loop, and cultured with shaking in JCM26 medium (5 ml) at 30 ° C for 6 hours.

Main cell culture:

 The preculture solution lm 1 was inoculated into 5 ml of JCM26 medium (isonicotinic acid hydrazide 200 mg / 50 ml) and cultured at 30 ° C with shaking. After completion of the culture, it was cooled on ice. The culture was centrifuged at 15000 rpm for 10 minutes to recover the cells.

Plasmid extraction:

 A plasmid was extracted from the collected cell pellet using Ml] 3 I Kit (Qiagen). At the time of recovery, the bacterial cell pellet was suspended in 1 ml of P 1 buffer containing 6 mg of lysozyme and shaken for 1 hour in a 37 ° C water bath. ,

1-2. Restriction enzyme treatment and ligation

 In order to perform ligation with PHSG 398 (cut with B amH I), pCG 100 obtained in 1-1 was cleaved with the restriction enzyme B g 1 II, pHSG 398 was dephosphorylated, and self-ligated. Chillon was prevented from happening.

Ligase pCGl OO (cut with B g I II) and pHSG 398 (cut with B m H I) And introduced into E. coli.

 1— 3. Transformation

Transformation was performed by applying 2.5 kvZ 200 Ω / 25 μF to the TOP 10 electrified cells. The obtained E. coli transformant was inoculated with 0.5 ni U of SOC medium and placed in a 15 ml centrifuge tube. The culture was shaken at 37 ° C for 1 hour. 37 100 mu 1 spread LBCm culture destinations in ° C, at 30 ° C, and stationary culture was performed _ώ

 1 1 4. Check the insert

 Plus and medium were extracted from E. coli ^ transformants and the insert was confirmed (Fig. 1). When cleaved with the restriction enzyme Pvu I, three bands (3944 bp, 777 bp, and 560 bp) should be detected if a hybrid plasmid is constructed. In the sample No. 5, the expected three bands were detected with the calculated size, confirming the construction of the desired high and low deplasmids.

 (Example 2) Introduction of hybrid plasmid into coli Nebacterum dartamicum and confirmation of replication

 The Escherichia coli containing the hybrid plasmid obtained in Example 1 was cultured again to prepare a large amount of the plasmid, which was introduced into the Corynebacterium dartamicam ATCC 13032 strain.

 2-1. Corynepacteria. Creation of glutamicum combi-t cells

 Pre-culture:

A colony of Corynebacterium 'glutamicum ATCC 13032 strain was inoculated into one loop. Culturing was carried out for 16 hours at 300 rpm and 30 ° C. in 5 ml of LB medium containing 0.2 ml of 50% glucose. At the end of the culture, OD ₆₆₀ was measured.

 Main culture:

The preculture, Sakaguchi flask containing Ep o medium 5.0 m 1, was inoculated to OD _{6. 60} is 0.3. Incubation was performed at 120 rpm for 18 hours.

 Cell recovery:

The OD _66Q was measured, and the culture was sufficiently cooled on ice. The culture solution was centrifuged at 15000 rpm for 10 minutes to recover the cells.

 Washing:

The obtained cells were suspended in 5 ml of 10% glycerin and cooled on ice. Centrifuge at 15000 rpm for 10 minutes and discard the supernatant. Pipetting with 10% glycerin in lm 1 And transferred to a 1.5 ml sterilized Eppen. Centrifugation was performed at 1 500 rpm for 1 minute, and the supernatant was discarded. Pipetted again with 1 ml of 10% glycerin. Centrifugation was carried out at 1 500 rpm for 1 minute, and the supernatant was discarded (4 times).

'Save:

 Pipetted with 0.5 m I of 10% glycerin and dispensed into 0.5 m 1 sterilized Eppen, 100 μl each. Quenched with liquid nitrogen and stored at 180 ° C.

 2-2. Transformation

 A voltage was applied at 2.5 k.v / 600 Ω / 25 μF to the competent cell obtained in 2-1. It was transferred to BH I S medium l rn 1 and placed in a 15 m 1 centrifuge tube. 4 Restoration culture was performed by allowing to stand in a 6 ° C water bath for 6 minutes, ice-cooling for 3 minutes, and then shaking culture at 30 ° C for 1 hour. L BHI IS medium Cm 30 and 60 were each plated with 5 plates in 2 sets for a total of 20 plates and statically cultured at 30 ° C.

 JCM 26 medium, Epo medium, BH I S medium and L BH I S plate were prepared as follows.

 JCM 26 medium composition:

 pH 7.2

E o medium:

 'The following B was prepared immediately before use and mixed with A to make 1 L.

A (B a c t0 tryptone 10 g,: B a c t o yeast extract 5 g, N a C l 10 g, N a C l 10 g, distilled water 80 m (autoclave sterilization))

B (Isonicotinohydrazide 4 g, Glycine 25 g, Twe e 8 0 1 ml, Distilled wood 200 ml (filter sterilized))

BH IS medium: The following A and B were mixed immediately before use.

A. (Nissui Pharmaceutical Brain Heart Incubation Powder 18.5 g, Distilled Water 800 ml

(Autoclave sterilization)

B (91 g sorbitol, 200 ml distilled water (autoclaved))

LBH I S medium:

 'The following A and B were mixed and spread on a plate. .

 A (B a c t to tryptone 5 g,: B a c t o yeast extract 2.5 g, NaC l 5 g, agar 18.5 g, distilled water 8 h 0 ml (autoclave-sterilized))

 B (91 g sorbitol, 200 ml distilled water (autoclaved))

2— 3. Confirmation of introduction of hybrid plasmid into Corynebaterum dartamicam Colonies grown on plates were inoculated into 3 ml of JCM26 Cm 60 medium and cultured at 30 ° C for 72 hours. The cells were collected by centrifugation and blastamide was extracted. In order to confirm whether the extracted plasmid was pCG100-pHSG3.98 hybrid plasmid (5280 bp), it was cleaved with the restriction enzyme KpηI.

 When confirmed by electrophoresis, a band of pCG100—pHSG398 (5280 bp) was confirmed at a position of 500 ° bp (Fig. 2).

 The constructed hybrid plasmid pCG100-pHSG 398 was confirmed to be stably retained in both E. coli and Corynebacterium dartamicam as a shuttle vector.

Cloning of g 1 _P FK gene (Example 3) Escherichia coli

 Escherichia coli has (a) glycerol uptake protein (glycerol facilitator) and (b) glycerol kinase as operon (g 1 p FK operon) as glycerin utilization gene, and in the order of promoter, facilitator, kinase. Arranged.

 A PCR was prepared from the fragment of promoter + (a) + (b) using the following primers, inserted into the pCG100_pHSG398 shuttle vector, and introduced into Corynebata glutamicum.

g 1 p— F K— F:

5, -ccccctgcaggcaacagagtgatggtatcaatc-3 '(P st I) (Guide (J number 5) g 1 p -FK-R 2:

5 '-ccccccctctagaaagcagcctttatcgcctactg-3' (X ba I) (酉 歹¹ J number 6)

 PCR amplification using KOD—gene polymerase yielded a band around 3350 bp (Figure 3), which was consistent with the size of g 1 p FK. This band was excised and recovered, and p CR4TOPO (Fig. 4).

 After cutting plasmids 1, 2, 5, 6, 7, 9, 10 with P st I—Xba I, target size inserts were confirmed by electrophoresis. The band was cut out and collected. Similarly, p CG100—pHSG398 ZBAP digested with P st I -X ba I and the previously excised fragment were ligated and introduced into large S. gonorrhoeae to obtain g 1 p FK—p CG 100—p HS G 398 plasmid. Obtained.

 g 1 p FK— pCG l 00— The pHSG398 plasmid was confirmed to contain 5 fragments of 1, 6, 8, 9 and 10 (Fig. 5). . A large number of plasmid No. 10 was prepared and introduced into Corynepacteria d'artamicam ATCC 13032 strain.

(Example 4) Introduction of g 1 _P FK gene into Corynebacterium glutamicum

 The plasmid g1pFK—pCGlOO—pHSG398, in which the Escherichia coli g1pFK gene was introduced into the pCGl00-pHSG398 shuttle vector, was introduced into the Corynebacterium glutamicum ATCC 13032 strain by electoral position. Since colony growth was confirmed on the LBCm60 p pm plate, the grown clones were cultured, and after plasmid extraction, the insert was confirmed. As a result, all of the 5 strains were found to have a band that seems to be g 1 p FK—p CG 10 O—p HSG 398 (FIG. 6).

 The resulting 5 strains of Corynebacterium glutamicum were inoculated into a medium containing glycerin as a carbon source30. When cultured overnight in C, all five strains showed good growth. From this result, it was clarified that the g 1 pFK gene derived from E. coli can function in Corynebacterium dartamicam cells and impart glycerin assimilation ability to Corynebacterium dartamicam.

 (Example 5) Confirmation of glycerin assimilation ability

The composition of the growth test medium was as follows. Urea 4 g

Ammonium sulfate 1 4 g

KH ₂ P0 ₄ 0.5 g

K ₂ HP0 ₄ 0.5 g

Mg S 0 ₄ 7H ₂ 0 0.5 g

Mn S 0 ₄ 7H ₂ 0 2 0 mg

F e S 0 ₄ 7H ₂ 0 2 0 mg

Piotin 2 0 0 μg

Thiamine hydrochloride 1 0 0 i g

1 g yeast extract

1 g casamino acid

p H 7.8 (KOH)

Distilled water L After adding the carbon source after autoclaving the above medium, 4 O ml each of filter-sterilized 50% glucose or 50% glycerin was added.

5-1. Growth test

Corynepacterium 'Dartamicam ATC C 1 3 0 3 2 Wild strain or g 1 p FK transgenic strain was inoculated from the plate into the growth test medium and grown at 30 ° C for 24 hours. ₆₆ . The turbidity of was measured. The results are shown in the table below. Carbon source Glucose Glycerin None

Wild strain 15.3 0.85 0.87

glpFK-introduced strain 14.8 13.9 0.69

Strain introduced only with plasmid 15.8 0.74 0.80 The above results indicate that the wild-type Corynebacterium 'glutamicum strain does not have glycerin-utilizing ability, whereas the recombinant bacterium of the present invention has glycerin-utilizing ability. It was done. Moreover, it turns out that the recombinant bacteria of this invention have the glycerol utilization ability equivalent to glucose utilization ability.

5— 2. Growth test of bacteria classified as Corynebacterium dartamicum

The following wild strains classified as Corynebacterium Riu arm glutamicum, when the same manner as described above glucose or glycerin as a carbon source, or the growth of the time without a carbon source 3 0 in cultured for 24 hours, OD ₆₆₀ Was tested by measuring the turbidity.

 (1) Corynepacterum dartamicum ATC C 1 3 0 3 2 (NB RC 1 2 1 6 8)

(2) Corynebacterium efficiens J CM 1 1 1 8 9 (3) Corynebacterium gnoretamicam AT CC 1 3 8 6 9

 (.4) Brevibacterium ammoniagenes

 C 1 2 0 7 2

 (5) Brevipacterium 'Ammonia Genes NB R C 1 2 6 1 2

The results are shown in the table below.

 As mentioned above, the wild strains of les were also vigorously growing when dulcose was used as the carbon source, but when Dali serine was used as the carbon source, there was a difference from the condition without carbon source. There wasn't. Therefore, it was confirmed that the wild strain classified as Corynebacterium dartamicam has no glycerin assimilation ability.

 (Example 6) Production of amino acids

 The following medium was placed in a 2 L jar fermenter, autoclaved, and cultured in 50 ml of the same composition. Escherichia coli g 1 p FK gene introduced Corynepactum glutamicum ATC C 1 3 0 3 2 The strain was inoculated and cultured at 30 ° C. for 48 hours while adjusting the pH to 7.2 with 25% aqueous ammonia while aeration was performed at 0.5 vvm.

 Medium composition:

 Glycerin 1 0 0 g

 Ammonium sulfate 1 0 g

KH ₂ P 0 ₄ 2 g

Mg S 0 ₄ 7H ₂ 0 1

F e S 0 ₄ 1 0 mg

Mn SO ₄ 1 0 mg

 Thiamine hydrochloride: 0.5 mg

 Biotin 5 μg

 Flavor 1 0m 1

 After culturing, the cells were removed by centrifugation to obtain a supernatant. When the culture broth was analyzed by high performance liquid chromatography, 6 g of glutamic acid was contained in 1 L.

- How, when subjected to the same culture with _§ 1 p FK gene without a introduces Corynebacterium Riu-time Darutamikamu ATC C 1 3 0 3 2 strains of colon ¾, cell growth has failed is little

(Example 7) Production of organic acid Culture medium 10 Om 1 is sterilized in a 500 ml 1 flask, sterilized 50% glycerol solution 4 ml 1 is added, and E. coli g 1 p FK gene introduced Corynebacterium glutamicum AT CC 13032 strain is inoculated, 30 ° Incubated with C for 24 hours (1 Sakaguchi). After completion of the culture, the cells were centrifuged. Wet cells obtained from 100 ml of culture broth were 3.7'3 g. This was used for the reaction.

 Add 100 ml of the reaction medium to a 100 ml medium bottle, add the bacterial cells and 12 ml of 50% glycerin solution (work in the glove box), and keep the reaction sealed at 30 ° C for 24 hours with stirring with a stirrer. I let you. After the reaction, the culture solution was centrifuged and analyzed.

 Analysis was performed using an HPLC ion exchange column KC-81 1 ODS. As a result, the pH after 24 hours of reaction was 5.7. The culture broth contained malic acid 0.4%, succinic acid 1.4%, acetic acid 0.2%, and lactic acid '1.9%.

 On the other hand, when the same culture was carried out using Corynebacterium dartamicum ATCC 13032 strain into which the Escherichia coli g 1 p FK gene was not introduced, the cells did not grow.

 The results of Examples 6 and 7 indicate that amino acids and organic acids can be produced by culturing a recombinant bacterium introduced with a gene encoding a glycerin uptake protein in a medium containing glycerin:! It was.

 (Example 8) Giving glycerin assimilation ability to Ralstonia eutropha ATCC 176.97 strain

 Ralstonia eutropha ATCC 17697 cannot grow using glycerin as a carbon source. Glycerol-utilizing ability was conferred by introducing the glycerol-utilizing genes g 1 pF and g 1 pK derived from Escherichia coli into this Ralstonia eutropha ATCC 17697 strain.

 8-1.Preparation of g 1 p F K gene fragment

 The g 1 p FK gene fragment was amplified by PCR using the following primer set using the g 1 p FK / p CG 100-pHSG 398 plasmid introduced into Corynebata d'artamicam as a saddle type.

g 1 pFK— F: 5 -ccccccccatggcaacagagtgatggtatcaatc-3 '(Self number 13)

 N c o I site

g 1 p F — R 2: 5 -ccccccccatggaagcagcctttatcgcctactg-3 (酉 G column number 14

PCR amplified a 3.3 kbp g 1 p FK gene fragment. It was fixed on a '4 TO PO cloning vector to obtain g 1 p FK-Nco I / pCR4TOPO plasmid. This plasmid was set as a PCR primer region, cleaved at the restriction enzyme site NcoI, separated and stained by electrophoresis, and then a 3.3 kbp fragment was excised and collected.

 8— 2. Vector preparation and ligation

 The broad host vector pBBR122 plasmid (MoB i Tec) contains resistance genes for two antibiotics, chloramphenicol and kanamycin. In order to introduce the g 1 p FK gene fragment into the restriction enzyme N co I site existing on this chloramfu unicol resistance gene, it was cleaved with the restriction enzyme Nco I, then treated with alkaline phosphatase and dephosphorylated. did. This vector DNA fragment was separated by electrophoresis, cut out and recovered. This fragment and the previous g 1 p FK gene fragment were ligated and introduced into E. coli to obtain g 1 p FKZp BBR122 plasmid.

 When the insert of g 1 pFKZpBBRl 22 plasmid- was confirmed, an insert of 3.3 k bp and a vector of 5.3 k bp could be confirmed. This plasmid was prepared in large quantities and introduced into Ralstonia 'Utropha ATCC 17697 strain.

 8-3. Creation of Ralstonia 'Utropha ATCC 17697 strain competent cell Ralstonia. Colony of Utropha ATCC 17697 strain was inoculated into 5 ml of LB medium in 1 loop, and cultured overnight at 300 rpm and 30 ° C. . 0.5 ml of the culture solution was planted in 50 ml of LB medium, cultured at 30 ° C, '120 rpm for 3 hours, and the culture solution was sufficiently cooled on ice. The culture was centrifuged at 15000 rpm for 10 minutes to recover the cells. The obtained cells were suspended in 5 ml of sterilized 10% glycerin and cooled on ice. Centrifuge for 10 minutes at 1.5000 rpm and discard the supernatant. Pipeted with 10% glycerin of lm 1 and transferred to a 1.5 ml sterilized Eppen. Centrifuge at 15000 rpm for 1 minute and discard the supernatant. Pipet again with lm 1 10% glycerin, centrifuge at 15000 for 1 min and discard supernatant. This operation was repeated 4 times. The obtained cells were suspended in 0.5 ml of 10% glycerin to obtain a competent cell.

 8-4. Introduction of g 1 p FK gene to Ralstonia 'Utropha ATCC 17697 strain Λ

Plasmid g 1 p F in which Escherichia coli g 1 p FK gene is introduced into pBBR122 plasmid The KpBBR122 plasmid was introduced into the Lanorestonia utropha ATC C 17697 strain by electroporation. Add 2 μ 1 of g 1 p FK / p B BR 122 plasmid solution (1.5 μg / μ 1) prepared in large quantities to the previously prepared combitent cell 50 μ 1, 2.5 KV, 250 Ω, A voltage was applied at 25 μF.

In the same manner, a voltage was applied to a solution in which 2 μl of pBBR 12 2 plasmid solution without insert was added instead of gl pFK / pBBR122 plasmid solution. After voltage application, the suspension was suspended in 1 ml of SOC medium, and cultured with shaking at 30 for 1 hour. After Γ time, the entire amount was inoculated into 50 ml of medium supplemented with 50 ppm of kanamycin in NB medium and cultured overnight at 3 CL ° C. After one night, the culture solution was diluted to 10 ³ to 10 ⁶ , spread on a medium obtained by adding 300 ppm of kanamycin to NB medium, and statically cultured at 30 ° C. to form colonies.

 The grown colonies were inoculated into a medium obtained by adding 50 ppm of kanamycin to NB medium, and cultured at 30 for 10 hours. The cells were collected from 1.5 ml of the culture solution by centrifugation, and the plasmid was extracted. In order to confirm the extracted plasmid, it was cleaved with the restriction enzyme NcoI. When confirmed by electrophoresis, it was confirmed that the transformant with gl pFKZpBBR122 plasmid introduced was found to have bands derived from g 1 p FK and p BBR 122 plasmid at positions 5300 bp and 3300 bp. In the transformant into which BBR 122 plasmid was introduced, only a band of a size considered to be derived from the pBBR 122 plasmid could be confirmed at the 5300 bp position.

 The Ralstonia eutropha transformants were named Ralstonia eutropha / g 1 p FK / p B BR 122 and Ralstonia eutropha ZpBBRl 22, respectively. 8-5. Growth test of Ralstonia eutropha 'transformant

Ralstonia 'Utropha ATCC 17697 strain can be grown by using 3 strains of Ralstonia eutropha ATCC 17697 wild strain, the previous Lars small nya eutrophano _g 1 p FK / p B BR 122 and ralstonia eutropha ZpBBRl 2 2 Growth was confirmed using a minimal medium containing L-malic acid as a single carbon source and a minimal medium containing glycerin as a single carbon source.

Ralstonia eutropha ATCC 17697 wild strain is NB medium 5 ml, ralstonia 'eutropha / g 1 p FK / p B BR 122 strain and Ralstonia eutropha / B BR 122 strain kanamycin 300 pm on NB medium Inoculated in 5 ml of the added medium and cultured overnight at 30 ° C. 50 μl of an overnight culture was inoculated into 50 ml of a medium supplemented with L-malic acid and glycerin as a carbon source in the following minimal medium, and cultured at 30 ° C. for 24 hours.

Growth after 24 hours incubation indicated by turbidity of OD ₆ 60 of the culture. Carbon source L-Malic acid Glycerin None

Wild strain 8.8 '0.07 0.06

glpFK strain 8.5 5.4 0.05

Strain introduced only with plasmid 9.2 0.05 0.07 From the above results, it is shown that the wild-type ralstonia utoguchifa has no glycerin assimilation ability, whereas the recombinant bacterium of the present invention has glycerin assimilation ability. It was. It can also be seen that the recombinant bacterium of the present invention has glycerin assimilation ability equivalent to L-malate assimilation ability in the wild strain.

 'The composition of the medium used in this example is as follows.

Minimal medium for growth test:

N a ₂ HP0 ₄ 12H ₂ 0 .9 g

KH ₂ P0 ₄ 0 .1 5 g

NH ₄ C 1 0. 0 5 g

Mg S 0 ₄ 7H ₂ 0 .2 g

 Trace metal solution 1 m 1

 Distilled water 1 L

 pH 7.0

Trace metal solution:

F e C.1 ₃ 9. 7 g

 C a C 1 a 7.8 g

C u S 0 ₄ 5H ₂ 0 0. 1 5 6 g

N i C 1 a 6H ₂ 0 0. 1 1 8 g

 Distilled water

 LB medium composition:

 Bacto Trypton

Bacto yeast extract Salt 10 g

 Distilled water 1 L

 Add 18 g of agar to the agar medium

S OC medium composition:

 Bacto Tryptone 20 g

 Bacto yeast extract 5 g

 Salt 0.5 g

 Distilled water 1 L

After autoclave sterilization 1M Mg CI ₂ solution sterilized by filtration as above 10ml, 1M

The mg S 0 ₄ solution 1 Om 1, 1M glucose solution 2 Om 1 was added.

NB medium composition:

 Bacto meat extract 3 g

 Bacto peptone 5 g

 Distilled water 1 L

 Add 18 g of agar to the agar medium

Example 9 Giving glycerin assimilation ability to Alcaligenes faecalis ATCC 18750

 Alkaline Genes faecalis ATCC 18750 cannot grow T using glycerin as a carbon source. The glycerin utilization genes g 1 pF and g 1 pK derived from E. coli were introduced into the Alkaligenes' Faecaris ATCC 18750 strain to impart the ability to assimilate dalyserin.

 Using the glpFK-pBBR122 plasmid used in Example 8, plasmid introduction and growth tests were similarly performed. As a transformant, Al force Ligenes fae force squirrel Zg 1 p F K / p BBR 122 and Al force Ligenes fae force squirrel / p BBR 122 were obtained.

 Al force Ligenes faecaris ATCC 18750 Wild strain and Al force Ligenes faecaris Zg 1 p FK / p B BR 122 and Al force Ligenes faecalis 7p BBR 1 22 Growth was confirmed on a minimal medium with L-malic acid as a single carbon source and a minimal medium with glycerin as a single carbon source.

Alkaline Genes' Faekaris ATCC 18750 wild strain is 5 ml in NB medium, Al Carigenes' faecaris Zg 1 p FK / p BBR 122 strain and Al force ligenes faye squirrel / p BBR 122 strain were inoculated into 5 ml of medium with 300 kpm of kanamycin added to NB medium, 30 ° C. overnight culture.

 50 1 of a medium supplemented with L-malic acid or glycerin as a carbon source in the following minimal medium was inoculated with 50 1 of the culture medium cultured overnight and cultured at 30 ° C. for 24 hours.

Growth after 24 hours of culture is OD ₆₆ of the culture. Of turbidity. Carbon source L-malic acid-glycerin None

Wild strain 12.3-0.06 0.04

glpFK-introduced strain 13.4 10.5 0.05

From the above results, it can be seen that the wild-type strain of Alcaligenes' Faecaris does not have glycerin assimilation ability, whereas the recombinant bacterium of the present invention has glycerin assimilation ability. Indicated. It can also be seen that the recombinant bacterium of the present invention has glycerin assimilation ability equivalent to L-malate assimilation ability in the wild strain. The composition of the minimal medium used for the growth test is as follows.

Minimal medium for growth test:

KH ₂ P0 ₄ 1 g

K ₂ HP0 ₄ 3 g

(H) ₂ S 0 ₄ 1 g

Mg S 0 ₄ 7H ₂ 0 0. 2 g

 Yeast extract 0.1 g

 1 liter of distilled water

 p H 6.8

 After autoclaving, 2.0 ml of a 50% carbon source solution sterilized by filtration was added and used. Example 1 0 Introduction of Corynebacterium diphtheria g 1 p A FK gene into Corynebacterium glutamicum

 1 0— 1. Preparation of g 1 p AF K gene fragment

 Corynebata diphtheria diphtheria ATCC 7009 71 D strain genomic DNA was transformed into a cocoon and the g 1 p A F K gene fragment was amplified with PCR.

D— g 1 p— F: 5 -cccccctctagatctcggcaccggtggtggtg-3 '(eyes cl 歹 U number 15)

C d— g 1 p— R: 5 '-ccccccctgcagcttggggaatacctgcgctgtcttc-3' (Self column number 1 6) A plasmid was extracted from the strain obtained by transformation, cut with PstI and XbaI, and an electrophoresis with a target size of 4600 bp was confirmed by electrophoresis. did. The shuttle vector pCG100_pHSG398 was double-strung with Xbal-PstI 'and electrophoresed, and then the shuttle vector band was cut out from the gel and recovered. This vector was ligated with the g 1 p AFK gene fragment excised from pCR40PO, and transformed into large intestine. The plasmid was recovered from the obtained transformant, cut with restriction enzymes XbaI and PstI, and the insert was confirmed. A 4600 bp band of the same size as the introduced g1p AF K gene was obtained. Was confirmed. This plasmid was named Cd_g 1 p AFK-pCG100-pHSG39 &.

 A combinatorial cell of Corynebatarum-Glutamicum was prepared and this plasmid was introduced by the electroporation method.

 10-2. Growth test of transformants

A growth test was conducted in the same manner as in Example 5 on the transformant and wild type strain obtained above. 'OD ₆₆ of the culture broth after 24 hours of culture. Of turbidity. Carbon source Glucose Glycerin None

 Wild strain 15.3 0.85 0.87

 glpAFK introduced strain 15.8 15.1 0.82

 Plasmid-only strain 15.8 0.74 0.80 The above results show that the wild-type strain of Corynebacterium dartamicam does not have glycerin-utilizing ability, whereas the recombinant bacterium of the present invention has glycerin-utilizing ability. It was done. In addition, it can be seen that the recombinant bacterium of the present invention has a glycerin assimilation ability equivalent to that in the wild strain.

 Industrial applicability

 According to the present invention, glycerol-assimilating ability can be imparted to bacteria. According to the present invention, means for effectively utilizing glycerin is provided.

 All publications, patents and patent applications cited in this specification shall be incorporated herein by reference as they are.

Claims

The scope of the claims

1. By introducing a glycerin uptake protein gene into a bacterium,

'How to grant phosphorus utilization ability.

 2. The method of claim 1, wherein the bacterium is a coryneform bacterium.

 3. The method according to claim 2, wherein the coryneform bacterium is Corynebataterum dartamicam.

 4. The method of claim 1 wherein the bacterium is a Ralstonia bacterium.

 5. The method according to claim 4, wherein the Ralstonia bacterium is Ralstonia utropha.

 6. The method according to claim 1, wherein the bacterium is an alkaligenus bacterium.

 7. The method according to claim 6, wherein the genus Alkagenes is Alkagenes faecalis.

 8. The method according to any one of claims 1 to 7, wherein the glycerin uptake protein gene is a g1p'F gene derived from E. coli.

 '9. The method according to any one of claims 1 to 7, wherein the glycerin uptake protein gene is a corynebacterium diphtheria-derived g 1 p F gene.

 10. The method according to any one of claims 1 to 9, wherein a glycerol kinase gene is further introduced into the bacterium.

 1 1. A recombinant bacterium having glycerin assimilation ability, into which a gene encoding a glycerin uptake protein has been introduced.

 12. The recombinant bacterium according to claim 11, which is a coryneform bacterium.

 13. The recombinant bacterium according to claim 12, which is Corynebacterium glutamicum.

14. The recombinant bacterium according to claim 11, which is a Ralstonia bacterium.

 15. The recombinant bacterium according to claim 14, which is Ralstonia 'utropha.

 16. The recombinant bacterium according to claim 11, which is a genus Alkagenes. '

 17. The recombinant bacterium according to claim 16, which is Alcaligenes faecalis.

18. The recombinant bacterium according to any one of claims 11 to 17, wherein the glycerin uptake protein gene is a g1pF gene derived from E. coli.

19. Glycerin uptake protein gene is derived from Corynebacterium diphtheria g 1 The recombinant bacterium according to any one of claims 11 to 17, which is a pF gene. 2. The recombinant bacterium according to any one of claims 11 to 19, further introduced with a glycerol kinase gene.

 2 1., The recombinant bacterium according to any one of claims 11 to 20 is cultured in a medium containing glycerin, and a fermentation product or a reaction product is obtained from the culture. A method for producing a fermentation product or a reaction product.