JP2011130680A - Method for producing sugar solution from residue of sugarcane-squeezed juice - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently saccharifying a residue of sugarcane-squeezed juice by smaller saccharification enzyme when saccharifying the residue. <P>SOLUTION: The sugar solution is obtained by separating sugarcane baggase into pith and rind, and reacting a saccharification enzyme such as cellulase and a mixture of the cellulase and hemicellulase, with the pith in a liquid to form sugar. The method for producing an objective substance such as an L-amino acid and a nucleic acid by a fermentation method using the obtained sugar solution or a fractionated product thereof as a carbon source is also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a technique for obtaining a sugar solution from sugarcane juice residue using a saccharifying enzyme.

Biomass, such as non-edible parts of plants such as sugarcane and corn, is expected to be used as a renewable energy source. For example, biomass ethanol produced from biomass is derived from carbon dioxide in the atmosphere fixed by photosynthesis, even if carbon dioxide is released into the atmosphere by its combustion, so the total amount of carbon in the air does not change The potential as an energy source is expected.
In order to use the above biomass as a fermentation substrate, it is necessary to treat polysaccharides such as cellulose, hemicellulose, and lignin, which are main components in the biomass solids, so that saccharifying enzymes possessed by microorganisms can be used. There are various techniques disclosed (Patent Documents 1 to 6 etc.). Moreover, the technique using the enzyme which mixed 2 or more types of cellulase and hemicellulase for the saccharification of woody biomass is also disclosed (patent document 7).

  The process of enzymatic saccharification of biomass consists of a pretreatment step (a treatment for making the saccharification enzyme easily act on cellulose and hemicellulose), and a saccharification step (a reaction in which saccharification enzyme is added to decompose cellulose and hemicellulose into monosaccharides) I know. Although it is technically possible to produce sugar by this process, it is not industrialized because of the high cost. Since the price of saccharifying enzymes is high, obtaining a sugar solution with a small amount of saccharifying enzymes was one problem for industrialization.

  Bagasse is a biomass produced as a by-product in large quantities when sugarcane is crushed and squeezed. Using this as a raw material, research and development for producing sugar, paper pulp, lacquer ware, board, feed, etc., and trial production and industrialization of products have been carried out. Techniques using bagasse as a fermentation raw material have also been proposed (Patent Documents 8, 9, etc.). As described above, in research on saccharification of bagasse, development research on an enzyme saccharification process that meets the target cost has been made by performing various pretreatments, but it has not been put into practical use.

  In addition, a technique is disclosed in which bagasse is separated into pis (okara-like medulla) and lined (fibrous hard structure), sugar is added to pis, lactic acid fermented, and paste is added to produce feed. (Patent Document 10) does not report saccharification of piss.

JP 2000-236899 A JP 2004-321174 A JP2009-59A JP2009-22165 JP 2009-155339 A Special table 2008-535664 JP2009-22165 JP2007-15881A Special table 2008-535524 JP-A-11-137183

  An object of the present invention is to provide a method of efficiently saccharifying with less saccharifying enzyme when saccharifying sugarcane juice residue.

  As a result of diligent research to solve the above problems, the present inventors have separated sugar cane bagasse into pis (oat-shaped medulla) and lined (epidermis), and saccharified the pis part more. The inventors have found that a sugar solution can be efficiently obtained with a small amount of saccharifying enzyme, and have completed the present invention.

That is, the present invention is as follows.
(1) A method for producing a sugar solution, comprising the step of reacting a pith separated from sugarcane with a saccharifying enzyme in a liquid to produce sugar.
(2) The method as described above, wherein the pis is separated from sugarcane bagasse.
(3) The method as described above, wherein the pis is obtained by separating sugar cane bagasse into pis and lined and collecting pis.
(4) The method as described above, wherein the saccharifying enzyme is cellulase or a mixture of cellulase and hemicellulase.
(5) A method for producing a target substance, wherein a sugar liquid is produced by the above method, and the target substance is produced by fermentation using the obtained sugar liquid or a fraction thereof as a carbon source.
(6) The method, wherein the target substance is an L-amino acid or a nucleic acid.

  According to the present invention, in order to obtain a sugar solution from sugarcane juice residue, it is possible to obtain a sugar solution with less saccharification enzyme, that is, at low cost. The obtained sugar liquid or a fraction thereof can be used as a raw material for ethanol fermentation or amino acid fermentation, and energy and useful substances can be produced from non-edible biomass.

Hereinafter, the present invention will be described in detail.
<1> Method for Producing Sugar Solution The present invention is a method for producing a sugar solution, comprising a step of reacting a pith separated from sugarcane with a saccharifying enzyme in a liquid to produce sugar.
“Saccharum” is a perennial herb that generally belongs to the family Gramineae, Panicoideae, Andropogoneae, Saccharum L., Saccharum spontaneum L., Saccharum officinarum L., Saccharum. Six species of robustum Jeswiet, Saccharum Barberi Jeswiet, Saccharum sinense Roxb., and Saccharum edule and their interspecific hybrids, but with closely related plants (Miscanthus genus, Sorghum genus, Erianthus genus, Ripidium genus, etc.) Includes intergeneric hybrids containing 5% by weight or more of ready-to-use sugar (sucrose) per raw sugarcane.

  Piss refers to cell wall components derived from soft tissue, including sugarcane juice (sugarcane juice) inside the sugarcane stem.

  Examples of piss include those separated from sugarcane bagasse. For example, by separating sugarcane bagasse into pis and lined, pis can be obtained (Japanese Patent Laid-Open No. 11-137183). Bagasse is obtained as a residue after squeezing sugarcane. Bagasse is sometimes called megasse. Usually, when sugar cane is squeezed, about 25% of the whole remains as a residue, ie bagasse. Usually, after sugarcane is pulverized until it becomes fibrous, it is squeezed with a mill, or the sugar solution is taken out by leaching with a diffuser and the residue becomes bagasse, but the method of squeezing is not limited thereto. Bagasse usually contains about 45% moisture, about 55% fiber, and a small amount of sugar.

  After bagasse is left as it is or dried, pis is obtained as oval medulla by removing the fibrous line using a sieve. As the vibrating sieve, a rotating sieve, a vibrating sieve, an inclined sieve, or the like having 5 to 40 mesh, preferably 10 to 20 mesh can be used. The lined is a cell wall component derived from the sugarcane stem epidermis tissue and means a fibrous plant tissue.

  Piss also uses sugar cane (clean cane), wax layer, lined and soup soup using a cane separation system (Anna Onaha et al., Okinawa Agriculture Vol.33 no.1 p.61-73). It can also be obtained as a residue by separating the soft tissue part and squeezing the soft tissue part.

  The “sugar cane squeezed residue” corresponds to bagasse when bagasse is used as a raw material, and to piss when pis obtained by a cane separation system or the like is used. In addition, the squeezed juice may remain in the juice residue.

  A sugar solution is obtained by reacting a saccharification enzyme in a liquid with the above-mentioned pith to produce sugar. An example of a saccharifying enzyme is cellulase. Also, a mixture of cellulase and other saccharifying enzymes can be used. An example of such a saccharifying enzyme is hemicellulase.

  Cellulase is an enzyme involved in the hydrolysis of (1 → 4) -β-glycosidic bonds. It is an endoglucanase (EC3.2.1.4) that cleaves amorphous cellulose from the inside of the molecule. Exoglucanase that releases cellobiose (cellobiohydrolase) (EC3.2.1.91) and exo-1,4-β-D-glucosidase (EC3.2.1.74) that produces glucose from the ends of cellobiose and cellooligosaccharide There is. Commercially available cellulases are sold as a mixture of cellulases having the aforementioned activity, and enzymes derived from Aspergillus niger and Trichoderma reesei are often used. Novozymes' Cellic Ctec and Genencor's Accellerase are cost-effective and typical saccharification enzymes. As other commercially available enzymes, there may be mentioned Meisserase from Meiji Seika Co., Ltd.

  Hemicellulase is a general term for enzymes involved in hydrolysis of glycosidic bonds contained in hemicellulose. Hemicellulose is a polysaccharide other than cellulose and pectin among the polysaccharides constituting the cell wall of land plant cells. The hemicellulose component contains a large amount of xylan, and the main component of hemicellulase is endo-1,4. -β-xylanase (EC 3.2.1.8), β-1,4-xylosidase (EC 3.2.1.37), etc., but other glucoside bond hydrolases are also included. Commercially available hemicellulases include Cellic Htec from Novozymes. They can also be obtained from Genencore and Amano Enzyme.

  When a saccharifying enzyme as described above is allowed to act on piss, glucose is mainly produced, but other sugars such as xylose, mannose, arabinose derived from hemicellose are also produced. These sugars may be further isomerized or decomposed by chemical reaction or enzymatic reaction depending on the application.

  Examples of the liquid used for the reaction include aqueous solvents such as a buffer solution. Depending on the enzyme used, a small amount of an organic solvent such as acetic acid may be contained. What is necessary is just to set reaction conditions, such as reaction temperature and pH suitably according to the description of the description attached to the commercially available enzyme, or by preliminary experiment etc. For example, when Cellic Ctec of Novozymes is used, conditions of 45 to 60 ° C. and pH 4.5 to 6.5 are mentioned. Examples of the enzyme amount include 2 to 6% (w / v). The reaction time is usually 24-48 hours.

  The reaction may be carried out by standing, but since the reaction between cellulose and hemicellulose and saccharifying enzyme is a solid-liquid reaction, it is preferably carried out with stirring.

  The pis can be used as it is as described above, but it may be dried once. Moreover, you may further pulverize piss.

  Prior to the enzyme reaction, pretreatment such as delignification or partial degradation of hemicellulose may be performed.

  The obtained sugar solution can be used as it is, or can be used as a dried product after removing moisture. Moreover, you may fractionate and use the component in a sugar liquid suitably. The fraction includes a crude product and a purified product. An example of the purified product is glucose.

<2> Method for producing target substance The sugar solution or fraction thereof obtained in the present invention can be used, for example, as a carbon source in the production of a target substance by fermentation. Examples of the target substance include alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and butanediol. Other examples include acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, succinic acid, citric acid, amino acid, and nucleic acid.

  In particular, L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, L-glycine, L-threonine L-serine, L-proline, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-cystine, L-methionine, L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine. Can be mentioned.

  The L-amino acid of the present invention also includes L-amino acid derivatives obtained using the above amino acids as starting materials, and examples include GABA, p-hydroxy-D-phenylglycine, DOPA, succinic acid, malic acid, and pyruvic acid. .

Examples of nucleic acids include purine nucleosides and purine nucleotides. Purine nucleosides include inosine, xanthosine, guanosine, adenosine, and the like, and purine nucleotides include 5'-phosphate esters of purine nucleosides, such as inosinic acid (inosine-5'-phosphate, hereinafter also referred to as "IMP"). Xanthylic acid (xanthosine-5′-phosphoric acid; hereinafter also referred to as “XMP”), guanylic acid (guanosine-5′-monophosphoric acid; hereinafter also referred to as “GMP”), adenylic acid (adenosine-5′-monophosphoric acid). (Hereinafter also referred to as “AMP”). The nucleic acid of the present invention also includes nucleic acid derivatives obtained using the above nucleic acid as a starting material, and examples include Ara-U (uracil arabinoside) and ZVA (Z-valacyclovir).
The target substance produced according to the present invention may be one type, or two or more types.

(2-1) Microorganism that can be used in the present invention The microorganism used in the present invention has a target substance-producing ability, and can accumulate the target substance in the medium or microbial cells when cultured in a liquid medium. If it is, it will not be restrict | limited in particular. The amount of the target substance to be accumulated may be any as long as it can be accumulated to the extent that it can be recovered from the medium.

  As the microorganism used in the present invention or a parent strain for breeding it, microorganisms belonging to the family Enterobacteriaceae such as Escherichia bacteria and Pantoea bacteria, coryneform bacteria, and the like can be used. Other microorganisms belonging to the family Enterobacteriaceae include Enterobacter, Klebsiella, Serratia, Erwinia, Salmonella, Morganella, etc. Enterobacteria belonging to γ-proteobacteria, and other microorganisms include Alicyclobacillus, Bacillus, yeast belonging to the genus Saccharomyces and Candida. It is done.

  Examples of Escherichia bacteria include those listed in the book by Neidhardt et al. (Neidhardt, FCet.al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington DC, 1208, table 1), for example, Escherichia coli it can. Examples of wild strains of Escherichia coli include the K12 strain or derivatives thereof, Escherichia coli MG1655 strain (ATCC No. 47076), and W3110 strain (ATCC No. 27325). To obtain these, for example, they can be sold from the American Type Culture Collection (ATCC) (address ATCC, Address: P.O. Box 1549, Manassas, VA 20108, 1, United States of America). That is, a corresponding registration number is assigned to each strain, and distribution can be received using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection (see http://www.atcc.org/).

  Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like, and Pantoea ananatis is an example of Pantoea bacteria. In recent years, Enterobacter agglomerans has been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc. by 16S rRNA nucleotide sequence analysis. There is something. In the present invention, any substance belonging to the genus Enterobacter or Pantoea may be used as long as it is classified into the family Enterobacteriaceae. Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and their derivatives when breeding Pantoea ananatis using genetic engineering techniques Can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans, but as described above, they have been reclassified as Pantoea Ananatis by 16S rRNA sequence analysis, etc. .

  Coryneform bacteria are a group of microorganisms defined in the Bergey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974), aerobic, Gram-positive, Non-acidic, microorganisms classified as gonococci having no sporulation ability can be used. Coryneform bacteria were previously classified as genus Brevibacterium but are now integrated as Corynebacterium (Int.J. Syst. Bacteriol., 41, 255 (1991)), and coryneform bacteria. Includes Brevibacterium and Microbatterium bacteria that are very closely related to the genus Bacteria.

Examples of such coryneform bacteria include the following.
Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lylium Corynebacterium merasecola Corynebacterium thermoaminogenes ( Corynebacterium efficiens
Corynebacterium herculis Brevibacterium divaricatam Brevibacterium flavum Brevibacterium inmariophilum Brevibacterium lactofermentum Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium Umm ammoniagenes Brevibacterium album Brevibacterium cerinum Microbacterium ammonia film

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
Corynebacterium lilium ATCC15990
Corynebacterium melasecola ATCC17965
Corynebacterium efficiens AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatam ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum ATCC13869 (Corynebacterium glutamicum ATCC13869)
Brevibacterium rose ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes ATCC6871, ATCC6872
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammonia film ATCC15354

  These can be obtained, for example, from the American Type Culture Collection (address P.O. Box 1549, Manassas, VA 2010812301 Parklawn Drive, Rockville, Maryland 20852, United States of America). In addition, AJ12340 shares were issued on October 27, 1987 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center) (Ibaraki, 305-8566, Japan) It is deposited under the Budapest Treaty under the accession number of FERM BP-1539 in 1st, 1st East, 1st Street, Tsukuba City. In addition, AJ12418 stock was deposited on January 5, 1989 at the Institute of Biotechnology, Ministry of International Trade and Industry under the contract number FERM BP-2205 under the Budapest Treaty.

When Bacillus bacteria are used, examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and the like.
Examples of Bacillus subtilis include Bacillus subtilis 168 Marburg (ATCC6051) and Bacillus subtilis PY79 (Plasmid, 1984, 12, 1-9), and examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T ( ATCC 23842), and Bacillus amyloliquefaciens N (ATCC 23845). Examples of Bacillus pumilus include Bacillus pumilus Gottheil No. 3218 (ATCC No. 21005) (US Pat. No. 3,616,206).

Hereinafter, a method for imparting L-amino acid or nucleic acid-producing ability to the parent strain as described above will be described.
In order to confer L-amino acid-producing ability, an auxotrophic mutant, an L-amino acid analog-resistant strain or a metabolically controlled mutant, and a recombinant strain with enhanced expression of an L-amino acid biosynthetic enzyme Can be applied to the breeding of amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (amino acid fermentation, Academic Publishing Center, Inc., May 30, 1986, first edition) Issue, see pages 77-100). Here, in the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation may be used singly or in combination of two or more. In addition, L-amino acid biosynthesis enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.

  In order to obtain an auxotrophic mutant, an analog-resistant mutant, or a metabolically controlled mutant having L-amino acid-producing ability, the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl. -N'-nitro-N-nitrosoguanidine and other mutants treated, etc. Among the obtained mutant strains, it shows auxotrophy, analog resistance, or metabolic control mutation, and has the ability to produce L-amino acid It can be obtained by selecting what it has.

  The L-amino acid-producing ability can also be imparted or enhanced by enhancing enzyme activity by gene recombination. Examples of the enhancement of enzyme activity include a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L-amino acid biosynthesis is enhanced. As a method for enhancing the expression of a gene, introducing an amplified plasmid in which a DNA fragment containing the gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication replication function of the plasmid in a microorganism, Alternatively, these genes can be achieved by making multiple copies on the chromosome by joining, transferring, etc., or by introducing mutations into the promoter regions of these genes (see International Publication No. 95/34672). .

  When the target genes are introduced onto the amplification plasmid or chromosome, the promoter for expressing these genes may be any promoter that functions in coryneform bacteria, and the promoter of the gene itself used. Or may be modified. The expression level of the gene can also be controlled by appropriately selecting a promoter that functions strongly in coryneform bacteria, or by bringing the -35 and -10 regions of the promoter closer to the consensus sequence. The method for enhancing the expression of the enzyme gene as described above is described in International Publication No. 00/18935, European Patent Application Publication No. 1010755, and the like.

  Hereinafter, a method for imparting L-amino acid-producing ability to bacteria and a bacterium imparted with L-amino acid-producing ability will be exemplified.

L-Threonine-Producing Bacteria Preferred microorganisms having L-threonine-producing ability include bacteria having enhanced activity of one or more of L-threonine biosynthetic enzymes. Examples of L-threonine biosynthesis enzymes include aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA) encoded by the thr operon, homoserine kinase (thrB), threonine synthase ( thrC), aspartate aminotransferase (aspartate transaminase) (aspC). The parentheses are abbreviations for the genes (the same applies to the following description). Of these enzymes, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase are particularly preferred. The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Escherichia in which threonine degradation is suppressed. Examples of the Escherichia bacterium in which threonine degradation is suppressed include, for example, the TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).

  The enzyme activity of the L-threonine biosynthetic enzyme is suppressed by the final product, L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is desirable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute the threonine operon, but the threonine operon forms an attenuator structure, and the expression of the threonine operon inhibits isoleucine and threonine in the culture medium. The expression is suppressed by attenuation. This modification can be achieved by removing the leader sequence or the attenuator of the attenuation region (Lynn, SP et al. 1987. J. Mol. Biol. 194: 59-69; WO 02/26993). Issue pamphlet; see International Publication No. 2005/049808 pamphlet).

  A unique promoter exists upstream of the threonine operon, but it may be replaced with a non-natural promoter (see WO98 / 04715), or the expression of a gene involved in threonine biosynthesis is expressed in lambda phage. A threonine operon as governed by a presser and promoter may be constructed. (See European Patent No. 0593792) In order to modify bacteria so that it is not subject to feedback inhibition by L-threonine, a strain resistant to α-amino-β-hydroxyvaleric acid (AHV) may be selected. Is possible.

  Thus, the threonine operon modified so as not to receive feedback inhibition by L-threonine has an increased copy number in the host or is linked to a strong promoter to improve the expression level. Is preferred. The increase in copy number can be achieved by transferring the threonine operon onto the genome by transposon, Mu-phage, etc., in addition to amplification by plasmid.

  In addition to the L-threonine biosynthetic enzyme, it is also preferable to enhance a glycolytic system, a TCA cycle, a gene related to the respiratory chain, a gene that controls gene expression, and a sugar uptake gene. Examples of these genes effective for L-threonine production include transhydronase (pntAB) gene (European Patent No. 733712), phosphoenolpyruvate carboxylase gene (pepC) (International Publication No. 95/06114 pamphlet), Phosphoenolpyruvate synthase gene (pps) (European Patent No. 877090), pyruvate carboxylase gene of Coryneform bacterium or Bacillus genus bacteria (International Publication No. 99/18228, European Patent Publication No. 1092776) Is mentioned.

  It is also preferable to enhance the expression of a gene imparting resistance to L-threonine and a gene conferring resistance to L-homoserine, or imparting L-threonine resistance and L-homoserine resistance to the host. Examples of genes that confer resistance include rhtA gene (Livshits, VA et al. 2003. Res. Microbiol. 154: 123-135), rhtB gene (European Patent Application Publication No. 0994190), rhtC gene (European patent application) Publication No. 1013765), yfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, the methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

  Examples of L-threonine producing bacteria or parent strains for deriving them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli FERM BP-3519 And FERM BP-3520 (U.S. Pat.No. 5,376,538), E. coli MG442 (Gusyatiner et al., 1978. Genetika (in Russian), 14: 947-956), E. coli VL643 and VL2055 (European Patent Application No. Strains belonging to the genus Escherichia, such as, but not limited to, 1149911).

  The TDH-6 strain lacks the thrC gene and is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. B-3996 shares were deposited with the accession number RIA 1867 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) on November 19, 1987 . This strain was also deposited with the Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on 7 April 1987 under the accession number B-3996. Has been.

  E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it. The B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned to Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318. Has been deposited internationally.

  The thrA gene encoding Escherichia coli aspartokinase homoserine dehydrogenase I is located at base numbers 337 to 2,799 on the genome sequence of Escherichia coli MG1655 registered under GenBank Accession No. U00096, and GenBank accession No. AAC73113 It is a gene encoding a protein registered in. The thrB gene encoding homoserine kinase of Escherichia coli is located at base numbers 2,801-3,733 on the genome sequence of Escherichia coli MG1655 strain registered in GenBank Accession No. U00096, and is registered by GenBank accession No. AAC73114 It is a gene that encodes the protein. The thrC gene encoding the threonine synthase of Escherichia coli is located at base numbers 3,734-5,020 on the genome sequence of Escherichia coli MG1655 strain registered in GenBank Accession No. U00096, and is registered under GenBank accession No. AAC73115 It is a gene that encodes the protein. These three genes are encoded as a threonine operon consisting of thrLABC downstream of the thrL gene encoding the leader peptide. In order to increase the expression of the threonine operon, it is effective to remove the attenuator region that affects transcription, preferably from the operon (WO 2005/049808, WO2003 / 097839).

  The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, which is resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.

  The rhtA gene has nucleotide numbers 848,433 to 849,320 on the genome sequence of Escherichia coli MG1655 strain registered under GenBank Accession No. U00096 acquired as a gene that gives resistance to homoserine and threonine (rht: resistant to threonine / homoserine). It is a gene encoding a protein located in (complementary strand) and registered in GenBank accession No. AAC73900. It has also been found that the rhtA23 mutation that improves the expression of rthA is a G → A substitution at position −1 relative to the ATG start codon (Livshits, VA et al. 2003. Res Microbiol. 154: 123- 135, European Patent Application No. 1013765).

  The asd gene of Escherichia coli is located at base numbers 3,571,798-3,572,901 (complementary strand) on the genome sequence of Escherichia coli MG1655 strain registered in GenBank Accession No. U00096, and is registered in GenBank accession No. AAC76458 It is a gene that encodes a protein. It can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene (see White, T. J. et al. 1989. Trends Genet. 5: 185-189.). The asd gene of other microorganisms can be obtained similarly.

  In addition, the aspC gene of Escherichia coli is located at base numbers 983, 742 to 984,932 (complementary strands) on the genome sequence of Escherichia coli MG1655 registered in GenBank Accession No. U00096, and is registered in GenBank accession No. AAC74014 It is a gene that encodes a protein that can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.

L-lysine-producing bacteria Hereinafter, L-lysine-producing bacteria and their construction methods are shown as examples.
For example, L-lysine analog resistant strains or metabolic control mutants can be mentioned as strains having L-lysine producing ability. Examples of L-lysine analogs include oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (hereinafter sometimes abbreviated as “AEC”), γ-methyllysine, α-chloro. Although caprolactam etc. are mentioned, it is not limited to these. Mutants having resistance to these lysine analogs can be obtained by subjecting bacteria belonging to the family Enterobacteriaceae or coryneform bacteria to ordinary artificial mutation treatment. Specific examples of L-lysine-producing bacteria include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see JP-A-56-18596 and US Pat. No. 4,346,170), Escherichia coli VL611. Strains (JP 2000-189180 A) and the like. In addition, WC196 strain (see International Publication No. 96/17930 pamphlet) can also be used as an L-lysine producing bacterium of Escherichia coli.

  An L-lysine producing bacterium can also be constructed by increasing the enzyme activity of the L-lysine biosynthesis system. These increases in enzyme activity can be achieved by increasing the copy number of the gene encoding the enzyme in the cell or by modifying the expression regulatory sequence.

  The modification for enhancing the expression of the gene can be performed, for example, by increasing the copy number of the gene in the cell using a gene recombination technique. For example, a DNA fragment containing the gapA gene may be ligated with a vector that functions in a host bacterium, preferably a multicopy vector, to produce a recombinant DNA, which is introduced into the bacterium and transformed.

  Increasing the copy number of a gene can also be achieved by having multiple copies of the above gene on the bacterial genomic DNA. In order to introduce a gene in multiple copies on bacterial genomic DNA, homologous recombination is performed using a sequence present in multiple copies on the genomic DNA as a target. As a sequence present in multiple copies on genomic DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. In addition, each gene may be linked in tandem beside the gapA gene present on the genome, or may be redundantly incorporated on an unnecessary gene on the genome. These gene introductions can be achieved using a temperature sensitive vector or using an integration vector.

  Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, a gene can be mounted on a transposon and transferred to introduce multiple copies onto genomic DNA. Confirmation of gene transfer on the genome can be confirmed by Southern hybridization using a part of the gene as a probe.

  Furthermore, in addition to the amplification of the gene copy number described above, the gene expression can be enhanced by the method described in the pamphlet of International Publication No. 00/18935 using expression control sequences such as each promoter of the gene on genomic DNA or plasmid. Regulators that replace powerful genes, bring the -35 and -10 regions of each gene closer to the consensus sequence, amplify regulators that increase gene expression, or reduce gene expression It can also be achieved by deleting or weakening. For example, lac promoter, trp promoter, trc promoter, tac promoter, araBA promoter, lambda phage PR promoter, PL promoter, tet promoter, T7 promoter, φ10 promoter and the like are known as strong promoters. It is also possible to introduce a base substitution or the like into the promoter region or SD region of the gapA gene and modify it to a stronger one. Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev. 1995. 1: 105-128) and the like. In addition, it is known that the substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, greatly affects the translation efficiency of mRNA, These can be modified. Expression regulatory regions such as gene promoters can also be determined using a promoter search vector or gene analysis software such as GENETYX. These promoter substitutions or modifications enhance gene expression. For example, a method using a temperature-sensitive plasmid or a Red driven integration method (WO2005 / 010175) can be used to replace the expression regulatory sequence.

  Examples of genes encoding L-lysine biosynthesis enzymes include dihydrodipicolinate synthase gene (dapA), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), and diaminopimelate decarboxylase gene (lysA). , Diaminopimelate dehydrogenase gene (ddh) (international publication No. 96/40934 pamphlet), phosphoenolpyruvate carboxylase gene (ppc) (Japanese Patent Laid-Open No. 60-87788), aspartate aminotransferase gene (aspC) ( Japanese Patent Publication No.6-102028), diaminopimelate epimerase gene (dapF) (Japanese Patent Laid-Open No. 2003-135066), aspartate semialdehyde dehydrogenase gene (asd) (WO 00/61723 pamphlet), etc. Gene of enzyme of acid pathway or homoaconite hydratase gene ( It includes genes such as enzymes of aminoadipic acid pathway Open Patent Laid-open No. 2000-157276) and the like. Further, the parent strain encodes a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Pat. No. 5,830,716), and a protein having L-lysine excretion activity. The expression level of the ybjE gene (WO2005 / 073390), the gene encoding glutamate dehydrogenase (gdhA) (Valle F. et al. 1983. Gene 23: 199-209), or any combination of these It may be. In parentheses are abbreviations for these genes.

  Wild-type dihydrodipicolinate synthase derived from Escherichia coli is known to undergo feedback inhibition by L-lysine, and wild-type aspartokinase derived from Escherichia coli undergoes inhibition and feedback inhibition by L-lysine. It has been known. Therefore, when using the dapA gene and the lysC gene, these genes are preferably mutant genes that are not subject to feedback inhibition by L-lysine.

  Examples of DNA encoding a mutant dihydrodipicolinate synthase that is not subject to feedback inhibition by L-lysine include DNA encoding a protein having a sequence in which the histidine residue at position 118 is substituted with a tyrosine residue. As a DNA encoding a mutant aspartokinase that is not subject to feedback inhibition by L-lysine, the threonine residue at position 352 is replaced with an isoleucine residue, the glycine residue at position 323 is replaced with an asparagine residue, 318 Examples include DNA encoding AKIII having a sequence in which the methionine at the position is replaced with isoleucine (see US Pat. Nos. 5,610,010 and 6,040,160 for these variants). Mutant DNA can be obtained by site-specific mutagenesis such as PCR.

  Wide host range plasmids RSFD80, pCAB1, and pCABD2 are known as plasmids containing mutant dapA encoding mutant mutant dihydrodipicolinate synthase and mutant lysC encoding mutant aspartokinase (USA) Patent No. 6040160). The Escherichia coli JM109 strain (US Pat. No. 6,040,160) transformed with this plasmid was named AJ12396, and this strain was established on 28 October 1993 at the Institute of Biotechnology, Ministry of International Trade and Industry (METI). Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) as deposit number FERM P-13936, transferred to an international deposit based on the Budapest Treaty on November 1, 1994, with the deposit number of FERM BP-4859 It is deposited with. RSFD80 can be obtained from AJ12396 strain by a known method.

  In the production of L-lysine, examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, and the like, and a strain in which the activity of the enzyme is reduced or deleted is disclosed in WO95 / 23864, It is described in WO96 / 17930 pamphlet, WO2005 / 010175 pamphlet and the like.

  In order to reduce or eliminate lysine decarboxylase activity, it is preferable to reduce the expression of both the cadA gene and ldcC gene encoding lysine decarboxylase. Decrease in the expression of both genes can be performed according to the method described in WO2006 / 078039 pamphlet.

  As a method for reducing or eliminating these enzyme activities, a mutation that reduces or eliminates the activity of the enzyme in the cell is applied to the gene of the enzyme on the genome by a usual mutation treatment method or gene recombination technique. What is necessary is just to introduce. Such mutations can be introduced, for example, by deleting a gene encoding an enzyme on the genome by genetic recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. Achieved. Also, introducing amino acid substitution (missense mutation) into the region encoding the enzyme on the genome, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation that adds or deletes one or two bases. It can also be achieved by deleting a part or the entire region of the gene (Wang, JP et al. 2006. J. Agric. Food Chem. 54: 9405-9410; Winkler, WC 2005. Curr. Opin. Chem. Biol. 9: 594-602; Qiu, Z. and Goodman, MF 1997. J. Biol. Chem. 272: 8611-8617; Wente, SR and Schachman, HK 1991. J. Biol. Chem. 266: 20833-20839 ). Also, construct a gene that encodes a mutant enzyme in which all or part of the coding region has been deleted, and replace the normal gene on the genome with the gene by homologous recombination, etc. By introducing it into the gene, the enzyme activity can be reduced or eliminated.

  For example, in order to introduce a mutation that reduces or eliminates the activity of the above enzyme by genetic recombination, the following method is used. A modified gene in which a partial sequence of the target gene is modified so that it does not produce a normally functioning enzyme is prepared, and a bacterium belonging to the family Enterobacteriaceae is transformed with the DNA containing the gene. By causing recombination with the above gene, the target gene on the genome can be replaced with a mutant. Such gene replacement using homologous recombination is a method called “Red-driven integration” (Datsenko, K. A, and Wanner, BL 2000. Proc. Natl. Acad. Sci. US A 97: 6640-6645), a combination method of Red driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ 2002. Bacteriol. 184: 5200-5203) (WO2005 / And the like, and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383; Japanese Patent Laid-Open No. 05-007491). Moreover, site-directed mutagenesis by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host.

  A preferred L-lysine-producing bacterium includes Escherichia coli WC196ΔcadAΔldcC / pCABD2 (WO2006 / 078039). This strain was constructed by disrupting the cadA and ldcC genes encoding lysine decarboxylase and introducing plasmid pCABD2 (US Pat. No. 6,040,160) containing a lysine biosynthesis gene from WC196 strain. The WC196 strain is a strain obtained from the W3110 strain derived from E. coli K-12, and encodes aspartokinase III in which feedback inhibition by L-lysine is released by replacing threonine at position 352 with isoleucine. After the wild type lysC gene on the chromosome of the W3110 strain was replaced with a mutant lysC gene (US Pat. No. 5,661,012), it was bred by conferring AEC resistance (US Pat. No. 5,827,698). The WC196 strain was named Escherichia coli AJ13069. On December 6, 1994, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center, Ibaraki, Japan, 305-8566, Japan Deposited as FERM P-14690 in Tsukuba City Higashi 1-chome 1 1 Central 6), transferred to the international deposit under the Budapest Treaty on 29 September 1995, and assigned the accession number FERM BP-5252 (US Pat. No. 5,827,698). WC196ΔcadAΔldcC itself is also a preferred L-lysine-producing bacterium. WC196ΔcadAΔldcC was named AJ110692 and was deposited internationally on October 7, 2008 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki, Japan 305-8566 Accession number FERM BP-11027 is assigned.

  pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation in which feedback inhibition by L-lysine is released, and a mutation in which feedback inhibition by L-lysine is released. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes (International Publication Nos. WO95 / 16042 and WO01 / 53459).

  The above-described methods for enhancing gene expression of enzymes involved in L-lysine biosynthesis and methods for reducing enzyme activity can be similarly applied to genes encoding other L-amino acid biosynthetic enzymes. .

  Examples of coryneform bacteria having L-lysine-producing ability include AEC-resistant mutant strains (Brevibacterium lactofermentum AJ11082 (NRRL B-11470), etc .: JP-B-56-1914, JP-B-56-1915 No. 57-14157, No. 57-14158, No. 57-30474, No. 58-10075, No. 59-4993, No. 61-35840, No. 62-24074, JP-B 62-36673, JP-B 5-11958, JP-B 7-112437, JP-B 7-112438); amino acids such as L-homoserine for its growth (See Japanese Patent Publication No. 48-28078, Japanese Patent Publication No. 56-6499); resistant to AEC, L-leucine, L-homoserine, L-proline, L-serine, L- Mutants that require amino acids such as arginine, L-alanine, and L-valine (see US Pat. Nos. 3,708,395 and 3,582,472) DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartate-analog, sulfa drugs, quinoids, L-lysine-producing mutants resistant to N-lauroylleucine; oxaloacetate decarboxylase (de) L-lysine production mutants exhibiting resistance to carboxylase) or respiratory enzyme inhibitors (JP 50-53588, JP 50-31093, JP 52-102498, JP 53) -9394, JP-A-53-86089, JP-A-55-9783, JP-A-55-9759, JP-A-56-32995, JP-A-56-39778, JP-B-53-43591, JP-B-53-1833); L-lysine production mutants requiring inositol or acetic acid (JP-A-55-9784, JP-A-56-8692); Fluoro L-lysine producing mutant strains sensitive to pyruvic acid or a temperature of 34 ° C. or higher (Japanese Patent Laid-Open No. 55-9783, JP-A-53-86090); Brevibacterium spp. Or Corynebacterium spp. Production mutants (US Pat. No. 4411997) which are resistant to ethylene glycol and produce L-lysine.

Examples of L-cysteine producing bacteria L-cysteine producing bacteria or parent strains for deriving them include E. coli JM15 (US Pat. No. 6,218,168) transformed with a different cysE allele encoding a feedback inhibition resistant serine acetyltransferase. , Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) having an overexpressed gene encoding a protein suitable for excretion of a substance toxic to cells, cysteine desulfohydrase activity E. coli strains such as reduced E. coli strains (JP-A-11-155571) and E. coli W3110 (international publication No. 0127307) with increased activity of transcription regulators of the positive cysteine regulon encoded by the cysB gene. Examples include, but are not limited to, the strains to which they belong.

Examples of L-leucine-producing bacteria L-leucine-producing bacteria or parent strains for inducing them include leucine-resistant E. coil strains (for example, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or β E. coli strains resistant to leucine analogs such as 2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, and 5,5,5-trifluoroleucine (Japanese Patent Publication No. 62-34397 and JP-A-8-70879), Although strains belonging to the genus Escherichia such as E. coli strains obtained by the genetic engineering method described in International Publication No. 96/06926, E. coli H-9068 (Japanese Patent Laid-Open No. 8-70879) can be mentioned, It is not limited to these.

  The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-leucine biosynthesis. As an example of such a gene, a gene of leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase preferably released from feedback inhibition by L-leucine is mentioned. . Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (European Patent Application Publication No. 1239041).

  L-isoleucine-producing bacteria of coryneform bacteria include coryneform bacteria (JP 2001-169788) in which a brnE gene encoding a branched-chain amino acid excretion protein is amplified, and L-isoleucine production by protoplast fusion with L-lysine-producing bacteria. Coryneform bacterium imparted with ability (JP-A 62-74293), coryneform bacterium with enhanced homoserine dehydrogenase (JP-A 62-91193), threonine hydroxamate resistant strain (JP 62-195293), α- Examples include ketomarone resistant strains (Japanese Patent Laid-Open No. 61-15695) and methyllysine resistant strains (Japanese Patent Laid-Open No. 61-15696).

Examples of L-histidine-producing bacteria L-histidine-producing bacteria or parent strains for inducing them include E. coli 24 strain (VKPM B-5945, Russian Patent No. 2003677), E. coli 80 strain (VKPM B- 7270, Russian patent 2119536), E. coli NRRL B-12116-B12121 (US Pat.No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US) Patent No. 6,344,347), E. coli H-9341 (FERM BP-6674) (European Patent Application Publication No. 1085087), E. coli AI80 / pFM201 (US Pat.No. 6,258,554), and other strains belonging to the genus Escherichia However, it is not limited to these.

  Examples of L-histidine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased. Examples of such genes include ATP phosphoribosyltransferase gene (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase gene (hisI), phosphoribosylformimino-5- Examples include aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (hisH), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), and histidinol dehydrogenase gene (hisD). It is done.

  L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine, and therefore L-histidine-producing ability is feedback-inhibited by the ATP phosphoribosyltransferase gene (hisG). Can be efficiently increased by introducing mutations that confer resistance to (Russian Patent Nos. 2003677 and 2119536).

  Specific examples of strains capable of producing L-histidine include E. coli FERM-P 5038 and 5048 into which a vector holding a DNA encoding an L-histidine biosynthetic enzyme has been introduced (Japanese Patent Laid-Open No. 56-005099). E. coli strain into which an amino acid transporting gene has been introduced (European Patent Application Publication No. 1016710), E. coli 80 to which resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin has been conferred Strains (VKPM B-7270, Russian Patent No. 2119536).

Examples of L-glutamic acid-producing bacteria L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, strains belonging to the genus Escherichia such as E. coli VL334thrC ⁺ (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K-12 strain (VKPM B-7). As a result, L-isoleucine-requiring L-glutamic acid producing bacterium VL334thrC ⁺ (VKPM B-8961) was obtained.

  Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, L-glutamic acid biosynthetic enzymes having one or two or more enhanced activities. Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), Methyl citrate synthase (prpC), phosphoenolpyruvate carbocilase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase ( eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate isomerase (tpiA), fructose bisphosphate aldolase ( fbp), phosphofructokinase (pfkA) , pfkB), glucose phosphate isomerase (pgi) and the like. Of these enzymes, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase are preferred.

  Examples of strains modified to increase expression of citrate synthetase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include European Patent Application Publication No. 1078989, European Patent Application Publication No. 955368. And those disclosed in European Patent Application No. 952221.

  As an example of an L-glutamic acid-producing bacterium or a parent strain for inducing the same, the activity of an enzyme that catalyzes the synthesis of a compound other than L-glutamic acid by diverging from the biosynthetic pathway of L-glutamic acid is reduced or deficient. Stocks are also mentioned. Examples of such enzymes include isocitrate triase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), Examples include acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), and the like. Bacteria belonging to the genus Escherichia lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity, and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. .

Specific examples include the following.
E. coli W3110sucA :: Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)

  E. coli W3110sucA :: Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter also referred to as “sucA gene”) of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.

In addition, examples of coryneform bacteria having reduced α-ketoglutarate dehydrogenase activity include the following strains.
Brevibacterium lactofermentum L30-2 strain (Japanese Unexamined Patent Publication No. 2006-340603)
Brevibacterium lactofermentum strain ΔS (International pamphlet No. 95/34672)
Brevibacterium lactofermentum AJ12821 (FERM BP-4172; see French patent publication 9401748)
Brevibacterium flavum AJ12822 (FERM BP-4173; French Patent Publication 9401748)
Corynebacterium glutamicum AJ12823 (FERM BP-4174; French Patent Publication 9401748)
Corynebacterium glutamicum L30-2 strain (Japanese Patent Laid-Open No. 2006-340603)

  Other examples of L-glutamic acid-producing bacteria include those belonging to the genus Escherichia and having resistance to an aspartic acid antimetabolite. These strains may be deficient in α-ketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5,908,768), and FFRM P- with reduced L-glutamate resolution. 12379 (US Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) and the like.

  An example of L-glutamic acid-producing bacteria of Pantoea ananatis is Pantoea ananatis AJ13355 strain. This strain is a strain isolated as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH from the soil of Kamata City, Shizuoka Prefecture. Pantoea Ananatis AJ13355 was commissioned on February 19, 1998 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Address: 1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki 305-8566, Japan) Deposited under the number FERM P-16644, transferred to an international deposit under the Budapest Treaty on 11 January 1999 and given the deposit number FERM BP-6614. The strain was identified as Enterobacter agglomerans at the time of its isolation and was deposited as Enterobacter agglomerans AJ13355, but in recent years, it has been identified by Pantoea ananatis (Pantoea ananatis) by 16S rRNA sequence analysis. ).

  In addition, examples of L-glutamic acid-producing bacteria of Pantoea ananatis include bacteria belonging to the genus Pantoea in which α-ketoglutarate dehydrogenase (αKGDH) activity is deficient or αKGDH activity is reduced. Such strains include AJ13356 (US Pat. No. 6,331,419) in which the αKGDH-E1 subunit gene (sucA) of AJ13355 strain is deleted, and sucA derived from SC17 strain selected from AJ13355 strain as a low mucus production mutant. There is SC17sucA (US Pat. No. 6,596,517) which is a gene-deficient strain. AJ13356 was founded on February 19, 1998, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome, 1-chome, Tsukuba, Ibaraki, Japan 305-8566 No. 6) was deposited under the deposit number FERM P-16645, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6616. AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification. The SC17sucA strain has been assigned a private number AJ417, and was deposited on February 26, 2004 at the above-mentioned National Institute of Advanced Industrial Science and Technology as the accession number FERM BP-08646.

  Furthermore, examples of L-glutamic acid-producing bacteria of Pantoea ananatis include SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain. The SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppsA), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain is a strain selected from this SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki 305-8566, Japan). Deposited as 17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the deposit number FERM BP-7207.

  Furthermore, as a method for conferring L-glutamic acid producing ability to coryneform bacteria, a method of amplifying the yggB gene encoding mechanosensitive channel (International Publication WO2006 / 070944), a mutation in which a mutation is introduced into the coding region It is also possible to use a method for introducing a type yggB gene. The yggB gene is located at base numbers 1,337,692 to 1,336,091 (complementary strand) on the genome sequence of Corynebacterium glutamicum ATCC 13032 strain registered under GenBank Accession No. NC_003450, and GenBank accession No. NP_600492, also called NCgl1221 It is a gene that encodes a registered membrane protein.

As another method for imparting or enhancing L-glutamic acid-producing ability, a method for imparting resistance to an organic acid analog or a respiratory inhibitor and a method for imparting sensitivity to a cell wall synthesis inhibitor may be mentioned. For example, a method of imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open No. 50-113209), a method of imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), and a method of weakening urease (Japanese Patent Laid-Open No. 52-038088) A method for imparting resistance to malonic acid (Japanese Patent Laid-Open No. 52-038088), a method for imparting resistance to benzopyrone or naphthoquinones (Japanese Patent Laid-Open No. 56-1889), a method of imparting HOQNO resistance (Japanese Patent Laid-Open No. 56-140895) ), A method for imparting resistance to α-ketomalonic acid (Japanese Patent Laid-Open No. 57-2689), a method for imparting resistance to guanidine (Japanese Patent Laid-Open No. 56-35981), a method for imparting sensitivity to penicillin (Japanese Patent Laid-Open No. 4-88994), etc. Is mentioned.
Specific examples of such resistant bacteria include the following strains.
Brevibacterium flavum AJ3949 (FERM BP-2632: see JP-A-50-113209)
Corynebacterium glutamicum AJ11628 (FERM P-5736; see JP 57-065198)
Brevibacterium flavum AJ11355 (FERM P-5007; see JP-A-56-1889)
Corynebacterium glutamicum AJ11368 (FERM P-5020; see JP 56-1889)
Brevibacterium flavum AJ11217 (FERM P-4318; see JP-A-57-2689)
Corynebacterium glutamicum AJ11218 (FERM P-4319; see JP-A-57-2689)
Brevibacterium flavum AJ11564 (FERM P-5472; see JP 56-140895 A)
Brevibacterium flavum AJ11439 (FERM P-5136; see JP-A-56-35981)
Corynebacterium glutamicum H7684 (FERM BP-3004; see JP 04-88994 A)
Brevibacterium lactofermentum AJ11426 (FERM P-5123; see JP-A-56-048890)
Corynebacterium glutamicum AJ11440 (FERM P-5137; see JP-A-56-048890)
Brevibacterium lactofermentum AJ11796 (FERM P-6402; see JP-A-58-158192)

Examples of L-phenylalanine producing bacteria L-phenylalanine producing bacteria or parent strains for deriving them include E. coli AJ12739 (tyrA :: Tn10, tyrR) lacking chorismate mutase-prefenate dehydrogenase and tyrosine repressor ( VKPM B-8197) (WO 03/044191), E. coli HW1089 (ATCC 55371) carrying a mutant pheA34 gene encoding chorismate mutase-prefenate dehydratase with desensitized feedback inhibition (US Pat.No. 5,354,672) Strains belonging to the genus Escherichia such as E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US Pat.No. 4,407,952). For example, but not limited to. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K that retains the gene encoding chorismate mutase-prefenate dehydratase whose feedback inhibition has been released. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 named AJ 12604 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579) can also be used (EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia having an increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publication Nos. 2003/0148473 and 2003/0157667, International Publication No. 03/044192). .

  Coryneform bacteria that produce phenylalanine include Corynebacterium glutamica BPS-13 strains (FERM BP-1777, K77 (FERM BP-2062) and K78 (FERM BP) with reduced phosphoenolpyruvate carboxylase or pyruvate kinase activity. -2063) (European Patent Publication No. 331145, JP 02-303495 A), tyrosine-requiring strain (JP 05-049489) and the like.

  In addition, as a phenylalanine-producing bacterium, by-modifying by-products into cells, for example, improving the expression level of L-tryptophan uptake genes tnaB, mtr and L-tyrosine uptake gene tyrP Also, a strain that efficiently produces L-phenylalanine can be obtained (EP1484410).

Examples of L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking tryptophanyl-tRNA synthetase encoded by the mutant trpS gene. (DSM10123) (U.S. Pat.No. 5,756,345), E. coli having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase not subject to feedback inhibition by tryptophan. SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614) E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Pat.No. 6,319,696) with increased ability to produce phosphoenolpyruvate Strains include belonging to Erihia genus, but is not limited thereto. L-tryptophan-producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 and 2003/0157667).

  Examples of L-tryptophan-producing bacteria or parent strains for inducing them include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), 3-deoxy-D-arabinohepturonic acid-7-phosphorus Acid synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolic acid pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC ), Prephenate dehydratase, chorismate mutase and tryptophan synthase (trpAB). One or more strains having enhanced activity are also included. Prefenate dehydratase and chorismate mutase are encoded by the pheA gene as a bifunctional enzyme (chorismate mutase / prephenate dehydrogenase (CM / PDH)). Among these enzymes, phosphoglycerate dehydrogenase, 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase, 3-dehydroquinate synthase, shikimate dehydratase, shikimate kinase, 5-enolic acid Pyruvylshikimate 3-phosphate synthase, chorismate synthase, prefenate dehydratase, chorismate mutase-prefenate dehydrogenase are particularly preferred. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase desensitized to feedback inhibition Examples include a transformant obtained by introducing plasmid pGH5 (International Publication No. 94/08031) into E. coli SV164.

  Examples of L-tryptophan-producing bacteria or parent strains for deriving them include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (JP-A 57-71397, JP-A 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO 2005/103275).

  Coryneform bacteria include Corynebacterium glutamicum AJ12118 (FERM BP-478 Patent 016881002), a coryneform bacterium introduced with a tryptophan operon (Japanese Patent Laid-Open No. 63240794), coryneform bacteria, which are resistant to sulfaguanidine. Coryneform bacteria (Japanese Patent Laid-Open No. 01994749) into which a gene encoding shikimate kinase derived therefrom has been introduced can be used.

Examples of L-proline-producing bacteria L-proline-producing bacteria or parent strains for deriving the same are E. coli 702ilvA (VKPM B-8012) that lacks the ilvA gene and can produce L-proline (European Patent Publication) Examples include, but are not limited to, strains belonging to the genus Escherichia such as No. 1,172,433).

  The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-proline biosynthesis. An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) encoding glutamate kinase that is desensitized to feedback inhibition by L-proline. Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (European Patent Publication No. 1,239,041).

  Examples of bacteria belonging to the genus Escherichia having L-proline producing ability include NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2000124295), German Patent No. 3127361 And E. coli strains such as those described in Bloom FR et al (The 15th Miami winter symposium, 1983, p. 34).

Examples of L-arginine producing bacteria L-arginine producing bacteria or parent strains for deriving them include E. coli 237 strain (VKPM B-7925) (US Patent Application Publication No. 2002/058315) and mutant N- Derived strains carrying acetylglutamate synthase (Russian Patent Application No. 2,001,112,869), E. coli 382 strain (VKPM B-7926) (European Patent Publication No.1,170,358), argA gene encoding N-acetylglutamate synthetase introduced Strains belonging to the genus Escherichia such as, but not limited to, arginine producing strains (European Patent Publication No. 1,170,361).

  Examples of L-arginine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased. Examples of such genes include N-acetylglutamylphosphate reductase gene (argC), ornithine acetyltransferase gene (argJ), N-acetylglutamate kinase gene (argB), acetylornithine transaminase gene (argD), ornithine carbamoyltransferase gene ( argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), carbamoylphosphate synthetase gene (carAB).

Examples of L-valine producing bacteria L-valine producing bacteria or parent strains for inducing the same include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). Not. It is preferable to remove the ilvGMEDA operon region required for attenuation so that the operon expression is not attenuated by the produced L-valine. Furthermore, it is preferred that the ilvA gene of the operon is disrupted and the threonine deaminase activity is reduced.
Examples of L-valine-producing bacteria or parent strains for deriving the same also include mutant strains having aminoacyl t-RNA synthetase mutations (US Pat. No. 5,658,766). For example, E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used. E. coli VL1970 was registered with Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on June 24, 1988 under the accession number VKPM B-4411. It has been deposited.
Furthermore, a mutant strain (International Publication No. 96/06926) that requires lipoic acid for growth and / or lacks H ⁺ -ATPase can be used as a parent strain.

  Examples of L-valine-producing bacteria of coryneform bacteria include strains modified so that expression of a gene encoding an enzyme involved in L-valic acid biosynthesis is enhanced. Examples of the enzyme involved in L-valinate biosynthesis include, for example, an enzyme encoded by the ilvBNC operon, that is, an acetohydroxyacid synthase encoded by ilvBN and an isomeroreductase encoded by ivlC (International Publication No. 00/50624) Is mentioned. Since the ilvBNC operon is regulated by operon expression by L-valine and / or L-isoleucine and / or L-leucine, the attenuation is released to release the suppression of the expression by L-valine produced. Is desirable.

  Coryneform bacteria having L-valine-producing ability may be performed by reducing or eliminating the activity of at least one enzyme involved in a substance metabolic pathway that reduces L-valine production. For example, it is conceivable to reduce the activity of threonine dehydratase involved in L-leucine synthesis and the enzyme involved in D-pantosenate synthesis (WO 00/50624).

Another method for imparting L-valine-producing ability includes a method for imparting resistance to amino acid analogs and the like.
For example, L-isoleucine and L-methionine auxotrophs, and mutant strains (FERM P-1841, FERM P having resistance to D-ribose, purine ribonucleoside or pyrimidine ribonucleoside and capable of producing L-valine) -29, JP-B 53-025034), mutants resistant to polyketoids (FERM P-1763, FERM P-1764, JP-B 06-065314), and in a medium containing acetic acid as the only carbon source. -Mutants (FERM BP-3006, FERM BP-3007, Patent 3006929) that are resistant to valine and that are sensitive to pyruvic acid analogs (fluoropyruvic acid, etc.) in a medium that uses glucose as the only carbon source. .

Examples of L-isoleucine-producing bacteria and L-isoleucine-producing bacteria or parent strains for inducing the same include mutants having resistance to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate Mutants having resistance to isoleucine analogs such as, and mutants having resistance to DL-ethionine and / or arginine hydroxamate (Japanese Patent Laid-Open No. 5-130882) are not limited thereto. Furthermore, a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).

  L-isoleucine-producing bacteria of coryneform bacteria include coryneform bacteria (JP 2001-169788) in which a brnE gene encoding a branched-chain amino acid excretion protein is amplified, and L-isoleucine production by protoplast fusion with L-lysine-producing bacteria. Coryneform bacterium imparted with ability (JP-A 62-74293), coryneform bacterium with enhanced homoserine dehydrogenase (JP-A 62-91193), threonine hydroxamate resistant strain (JP 62-195293), α- Examples include ketomarone resistant strains (Japanese Patent Laid-Open No. 61-15695) and methyllysine resistant strains (Japanese Patent Laid-Open No. 61-15696).

Examples of L-methionine-producing bacteria L-methionine-producing bacteria or parent strains for inducing them include, but are not limited to, L-threonine-requiring strains and mutants having resistance to norleucine (Japanese Patent Laid-Open No. 2000-2000). 139471). Furthermore, a strain lacking a methionine repressor, a recombinant strain transformed with a gene encoding a protein involved in L-methionine biosynthesis, such as homoserine transsuccinylase, cystathionine γ-synthase, can also be used as a parent strain. (Japanese Patent Laid-Open No. 2000-139471).

Next, a method for imparting nucleic acid-producing ability to a microorganism and a nucleic acid-producing bacterium will be exemplified.
Bacterial microorganisms having nucleic acid-producing ability can be obtained by, for example, imparting purine nucleoside requirements or resistance to drugs such as purine analogs to the above-described bacterial microorganisms (Japanese Patent Publication No. 38-23099). JP-B-54-17033, JP-B-55-45199, JP-B-57-14160, JP-B-57-41915, JP-B-59-42895). For example, Bacillus bacteria having auxotrophy and drug resistance are used for normal mutation treatments such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS (ethane methanesulfonate). It can be obtained by treatment with a mutagen.

Examples of Bacillus bacteria that produce purine nucleosides include the following.
As a specific example of the inosine-producing strain belonging to the genus Bacillus, the Bacillus subtilis KMBS16 strain can be used. The strain contains a deletion of purR gene encoding purine operon repressor (purR :: spc), deletion of purA gene encoding succinyl-AMP synthase (purA :: erm), and deoD gene encoding purine nucleoside phosphorylase. It is a known derivative of Bacillus subtilis trpC2 strain (168 Marburg) into which a deficiency (deoD :: kan) has been introduced (JP 2004-242610, US2004166575A1). Also, Bacillus subtilis strain AJ3772 (FERM P-2555) (Japanese Patent Laid-Open No. 62-014794) can be used.

  Examples of Bacillus bacteria having the ability to produce guanosine include Bacillus bacteria with increased IMP dehydrogenase activity (Japanese Patent Laid-Open No. 3-58787), vectors incorporating a purine analog resistance or decoinin resistance gene, and adenine-requiring mutants And Bacillus bacteria introduced in (4).

Examples of Bacillus bacteria that produce purine nucleotides include the following.
As inosinic acid-producing bacteria, inosine-producing strains with reduced phosphatase activity of Bacillus subtilis have been reported (Uchida, K. et al., Agr. Biol. Chem., 1961, 25, 804-805, Fujimoto, M. Uchida, K., Agr. Biol. Chem., 1965, 29, 249-259). As a guanylate-producing bacterium, it has adenine requirement, is resistant to decoinin or methionine sulfoxide, and can produce 5′-guanylic acid (guanosine-5′-monophosphate, hereinafter also referred to as “GMP”). And a mutant strain of the genus Bacillus (Japanese Patent Publication No. 56-12438).

  In addition, xanthylic acid-producing bacteria can be constructed using a method used for breeding coryneform bacteria centering on Corynebacterium ammmoniagenes. For example, by obtaining a PRPP amidotransferase-enhanced strain (Japanese Patent Laid-Open No. 8-168383), an aliphatic amino acid resistant strain (Japanese Patent Laid-Open No. 4-262790), a dehydroproline resistant strain (Korea Patent Publication No. 2003-56490), xanthylic acid production Fungi can be constructed.

  Moreover, the following method is mentioned as a method of breeding the Bacillus genus bacteria which have purine-type substance production ability. Examples include a method for increasing the intracellular activity of an enzyme involved in purine biosynthesis common to purine nucleosides and purine nucleotides, that is, purine biosynthesis enzyme. The intracellular activity of the enzyme is preferably increased as compared with an unmodified strain of Bacillus bacterium, for example, a wild-type Bacillus bacterium. “The activity increases” corresponds to, for example, a case where the number of enzyme molecules per cell increases or a case where the specific activity per enzyme molecule increases. For example, the activity can be increased by increasing the expression level of the enzyme gene. Examples of the enzyme involved in the purine biosynthesis include phosphoribosyl pyrophosphate amide transferase, phosphoribosyl pyrophosphate synthetase (PRPP synthetase [EC: 2.7.6.1]) and the like.

  A part of catabolite produced by metabolism by a sugar source such as glucose incorporated into the pentose phosphate system becomes ribose-5-phosphate via ribulose-5-phosphate. Biosynthesized ribose-5-phosphate produces purine nucleoside, histidine, and phosphoribosyl pyrophosphate (PRPP), an essential precursor for biosynthesis of tryptophan. Specifically, ribose-5-phosphate is converted to PRPP by phosphoribosyl pyrophosphate synthetase. Therefore, the ability to produce purine substances can be imparted to or enhanced in bacteria belonging to the genus Bacillus by modifying the activity of phosphoribosyl pyrophosphate synthetase to increase.

  “The activity of the phosphoribosyl pyrophosphate synthetase is increased” means that the activity of the phosphoribosyl pyrophosphate synthetase is increased relative to a non-modified strain such as a wild strain or a parent strain. The activity of phosphoribosyl pyrophosphate synthetase should be measured by, for example, the method of Switzer et al. (Methods Enzymol., 1978, 51, 3-11), the method of Roth et al. (Methods Enzymol., 1978, 51, 12-17) Can do. Bacteria belonging to the genus Bacillus having an increased activity of phosphoribosyl pyrophosphate synthase can be obtained by, for example, phosphoribosyl pyrophosphate by a method using a plasmid or a method of integrating on a chromosome in the same manner as described in JP-A-2004-242610. It can be produced by highly expressing a gene encoding a synthetase in a Bacillus bacterium.

  On the other hand, when PRPP, a precursor essential to purine nucleoside, histidine, and tryptophan biosynthesis, is produced, a part of it is converted into a purine nucleotide and purine nucleoside by a group of enzymes involved in purine biosynthesis. The genes encoding such enzyme groups include the Bacillus subtilis purine operon, specifically the purEKB-purC (orf) QLF-purMNH (J) -purD operon gene (Ebbole DJ and Zalkin H, J. Biol Chem., 1987, 262, 17, 8274-87 (now also called purEKBCSQLFMNHD: Bacillus subtilis and Its Closest Relatives, Editor in Chief: AL Sonenshein, ASM Press, Washington DC, 2002. GenBank Accession No. NC_000964) And the Escherichia coli pur regulon gene (Escherichia and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASM Press, Washington DC, 1996).

  Therefore, the ability to produce purine substances can be imparted or enhanced by enhancing the expression of these genes. The purine operon genes that can be used in the present invention are not limited to these genes, and genes derived from other microorganisms and animals and plants can also be used.

  Examples of a method for increasing the expression level of the purine operon include a method in which the purine operon gene is highly expressed in a Bacillus bacterium by a method using a plasmid or a method of integrating on a chromosome.

  As a second method for increasing the expression level of the purine operon, replacement of the promoter unique to the purine operon with a stronger promoter, or substitution of the -35 and -10 regions of the unique promoter with a consensus sequence can be mentioned. .

  For example, in Bacillus subtilis (Strain B. subtilis 168 Marburg; ATCC6051), the -35 sequence of the purine operon is a consensus sequence (TTGACA), but the -10 sequence is TAAGAT, which is different from the consensus sequence TATAAT ( Ebbole, DJ and H. Zalikn, J. Biol. Chem., 1987, 262, 8274-8287). Therefore, the transcriptional activity of the purine operon can be increased by making the -10 sequence (TAAGAT) into a consensus sequence, or by modifying it so that it becomes TATAAT, TATGAT, or TAAAAT close to the consensus sequence. The replacement of the promoter sequence can be performed by the same method as the gene replacement described below.

  As a third method of increasing the expression level of the purine operon, there is a method of decreasing the expression level of the purine operon repressor (USP 6,284,495). “Expression of the purine operon repressor” includes both transcription of the purine operon gene and translation of the transcript. Further, “reducing the expression level” includes that the expression level is lower than that in an unmodified strain, for example, a wild-type Bacillus bacterium, and that the expression is substantially lost.

  In order to reduce the expression level of the purine operon repressor (purine repressor), for example, the Bacillus bacterium is treated with an ultraviolet ray irradiation or a mutagen that is usually used for mutagenesis such as NTG or EMS. A method of selecting a mutant strain in which the expression level of the repressor is reduced can be employed.

  Further, Bacillus bacteria having a reduced expression level of the purine repressor may include, for example, a homologous recombination method using genetic recombination (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory press (1972); According to Matsuyama, S. and Mizushima, S., J. Bacteriol., 1985, 162, 1196-1202), a gene encoding a purine repressor on the chromosome (purR; GenBank Accession NC_000964 (the coding region has nucleotide numbers 54439 to 55293). ) With a gene that does not function normally (hereinafter sometimes referred to as a “disrupted gene”).

  Furthermore, the ability to produce purine substances can also be enhanced by weakening the incorporation of purine substances into cells. For example, purine nucleoside uptake into cells can be attenuated by blocking reactions involved in purine nucleoside uptake into cells. The reaction involved in the incorporation of the purine nucleoside into the cell is, for example, a reaction catalyzed by a nucleoside permease.

  Furthermore, when producing a purine nucleoside, the activity of an enzyme that degrades purine substances may be reduced in order to enhance the purine nucleoside production ability. Examples of such an enzyme include purine nucleoside phosphorylase.

  Purine nucleotides biosynthesized from PRPP by enzymes involved in purine biosynthesis are dephosphorylated and converted to purine nucleosides. In order to accumulate the purine nucleoside efficiently, it is preferable to further degrade the purine nucleoside to reduce the activity of purine nucleoside phosphorylase such as hypoxanthine. That is, it is desirable to modify so that the purine nucleoside phosphorylase using purine nucleosides such as inosine as a substrate is weakened or deleted.

  Specifically, reduction of purine nucleoside phosphorylase activity can be achieved by disrupting the deoD gene and pupG gene encoding purine nucleoside phosphorylase in Bacillus bacteria. The Bacillus bacterium of the present invention may be modified so as to destroy the deoD gene and the pupG gene as described above alone or simultaneously. As the deoD gene and pupG gene, for example, genes derived from the genus Bacillus (deoD; GenBank Accession No. NC_000964 (coding region is 2134672-2135370), pupG; GenBank Accession No. NC_000964 (coding region is 2445610-2446422) can be used.

  In addition, the activity of succinyl-AMP synthase may be decreased in order to enhance the purine substance production ability. An example of a gene encoding succinyl-AMP synthase is the purA gene. Examples of the purA gene include those having a base sequence registered under GenBank Accession No. NC — 000964 (the coding region is the complementary strand base number 4153460-4155749).

  Furthermore, the activity of inosine monophosphate (IMP) dehydrogenase may be reduced in order to enhance the ability to produce purine substances. Examples of the gene encoding IMP dehydrogenase include the guaB gene. Examples of the guaB gene include those having a base sequence registered in GenBank Accession No. NC — 000964 (coding region is 15913-17376).

 Further, as a method for enhancing the ability to produce purine substances, it is conceivable to amplify a gene that encodes a protein having an activity of discharging purine substances. Examples of the bacterium in which such a gene is amplified include a Bacillus bacterium in which the rhtA gene is amplified (Japanese Patent Laid-Open No. 2003-219876).

  When breeding the above-mentioned L-amino acid-producing bacterium by genetic recombination, the gene used is not limited to the gene having the genetic information described above or a gene having a known sequence, and the function of the encoded protein is impaired. Unless otherwise specified, genes having conservative mutations such as homologues and artificially modified variants of the genes can also be used. That is, it may be a gene encoding a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of a known protein.

  Here, “one or several” differs depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically, preferably 1 to 20, more preferably 1 to 10. Means, more preferably 1-5. A conservative mutation is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. Is an amino acid having a hydroxyl group between Gln and Asn, in the case of a basic amino acid, between Lys, Arg, and His, and in the case of an acidic amino acid, between Asp and Glu. In some cases, it is a mutation that substitutes between Ser and Thr. Typical conservative mutations are conservative substitutions. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys. , Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg Substitution, Glu to Gly, Asn, Gln, Lys or Asp substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe Substitution, Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Tyr substitution, Tyr to His, Phe or Trp substitution, and Val To Met, Ile or Leu It is below. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the microorganism from which the gene is derived. Also included by Such a gene can be modified, for example, by site-directed mutagenesis so that the amino acid residue at a specific site of the encoded protein contains substitutions, deletions, insertions or additions. Can be obtained by:

Furthermore, the gene having a conservative mutation as described above has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more with respect to the entire encoded amino acid sequence. And a gene encoding a protein having a function equivalent to that of a wild-type protein.
In addition, each codon in the gene sequence may be replaced with a codon that is easy to use in the host into which the gene is introduced.

  A gene having a conservative mutation may be one obtained by a method usually used for mutation treatment such as treatment with a mutation agent.

  A gene is a DNA that hybridizes with a probe complementary to a known gene sequence or a probe that can be prepared from the complementary sequence under stringent conditions and encodes a protein having a function equivalent to that of a known gene product. Also good. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. As an example, DNAs having high homology, for example, 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, are hybridized to each other. Conditions under which DNAs having low homology do not hybridize with each other, or washing conditions for normal Southern hybridization at 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably The conditions include washing once at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS, more preferably 2 to 3 times.

  As the probe, a part of the complementary sequence of the gene can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

(2-2) Microbial culture Using the sugar solution produced by the method of the present invention or a fraction thereof (hereinafter sometimes referred to as “sugar”) as a carbon source, the ability to produce wood low substances is obtained. The microorganisms are cultured, and the target substance is collected from the medium or microbial cells.
A method similar to a normal fermentation method can be used except that the sugar is used as a carbon source.

  The method of the present invention can use any of batch culture, fed-batch culture, and continuous culture, and the sugar obtained by the present invention is contained in the initial medium. It may be contained in the feed medium or in both of them.

  Fed-batch culture refers to a culture method in which a medium is fed continuously or intermittently into a culture container and the medium is not removed from the container until the end of the culture. Continuous culture refers to a method in which a medium is fed continuously or intermittently into a culture container and the medium (usually equivalent to the medium to be fed) is extracted from the container. The initial medium means a medium (medium at the start of the culture) used for batch culture (batch culture) before feeding the fed-batch medium in fed-batch culture or continuous culture. It means a medium supplied to a fermenter when fed-batch culture or continuous culture is performed. In addition, batch culture (batch culture) means a method in which a new medium is prepared every time, a strain is planted there, and no medium is added until harvest.

  The sugar may be used at any concentration that is suitable for producing the target substance. The sugar concentration in the medium is preferably about 0.05 w / v% to 50 w / v%, more preferably about 0.1 w / v% to 40 w / v%, particularly preferably 0.2 w / v% to 20 w / v. About% is contained in the medium. The sugar produced by the method of the present invention can be used alone or in combination with other carbon sources such as glucose obtained by other methods or fructose, sucrose, molasses, starch hydrolysate, etc. It can also be used. In this case, the sugar obtained by the method of the present invention and the other carbon source can be mixed at an arbitrary ratio. Preferred as other carbon sources are fructose, sucrose, lactose, galactose, molasses, starch hydrolysates, sugars such as sugar liquid obtained by hydrolysis of other biomass, alcohols such as ethanol and glycerol, Organic acids such as fumaric acid, citric acid and succinic acid.

  As the medium to be used, a medium conventionally used in the fermentation production of L-amino acids using microorganisms can be used except that the sugar obtained by the method of the present invention is used as a carbon source. That is, in addition to a carbon source, a normal medium containing a nitrogen source, inorganic ions, and other organic components as required can be used. Here, as the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, and urea, or organic nitrogen such as nitrate and soybean hydrolysate, ammonia gas, aqueous ammonia, and the like can be used. Further, peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean hydrolyzate and the like can also be used. Only 1 type of these nitrogen sources may be contained in the culture medium, and it may contain 2 or more types. These nitrogen sources can be used for both the initial medium and the fed-batch medium. In addition, the same nitrogen source may be used for both the initial culture medium and the feed medium, or a different nitrogen source from the initial culture medium may be used.

  The medium preferably contains a phosphate source and a sulfur source in addition to a carbon source and a nitrogen source. As the phosphoric acid source, phosphoric acid polymers such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate and pyrophosphoric acid can be used. The sulfur source may be any one containing sulfur atoms, but sulfates such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione are desirable. However, ammonium sulfate is desirable.

  In addition to the above components, the medium may contain a growth promoting factor (a nutrient having a growth promoting effect). Examples of the growth promoting factor include trace metals, amino acids, vitamins, nucleic acids, and peptone, casamino acid, yeast extract, soybean protein degradation products, and the like containing these. Examples of trace metals include iron, manganese, magnesium, calcium, and vitamins include vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12. These growth promoting factors may be contained in the initial culture medium or in the fed-batch medium.

  In addition, when using an auxotrophic mutant that requires an amino acid or the like for growth, the medium is preferably supplemented with the required nutrients. In particular, L-lysine-producing bacteria that can be used in the present invention have many L-lysine biosynthetic pathways as described later, and L-lysine resolution is weakened. -It is desirable to add 1 type (s) or 2 or more types chosen from homoserine, L-isoleucine, and L-methionine. The initial medium and fed-batch medium may have the same or different medium composition. The initial culture medium and the fed-batch medium may have the same or different sulfur concentration. Furthermore, when the feeding of the feeding medium is performed in multiple stages, the composition of each feeding medium may be the same or different.

  In addition, as long as the culture medium used by this invention is a culture medium containing a carbon source, a nitrogen source, and another component as needed, any of a natural culture medium and a synthetic culture medium may be sufficient.

  Cultivation is preferably carried out for 1 to 7 days under aerobic conditions, and the culture temperature is preferably 20 ° C to 45 ° C, preferably 24 ° C to 45 ° C, particularly preferably 33 to 42 ° C. The culture is preferably aeration culture, and the oxygen concentration is preferably adjusted to 5 to 50%, desirably about 10% with respect to the saturation concentration. Further, the pH during the culture is preferably 5-9. For adjusting the pH, inorganic or organic acidic or alkaline substances such as calcium carbonate, ammonia gas, aqueous ammonia and the like can be used.

  By culturing preferably for about 10 to 120 hours under the above conditions, a significant amount of the target substance is accumulated in the culture solution.

  In addition, when producing basic amino acids such as L-lysine, the pH during culture is controlled to 6.5 to 9.0, and the pH of the medium at the end of the culture is controlled to 7.2 to 9.0. Control the pressure to be positive, or supply carbon dioxide or a mixed gas containing carbon dioxide to the medium, and there is a culture period in which bicarbonate ions and / or carbonate ions in the medium are present at least 2 g / L or more. In addition, fermentation may be performed by a method in which the bicarbonate ion and / or carbonate ion is used as a counter ion of a cation mainly composed of a basic amino acid, and production may be performed by a method of recovering the target basic amino acid (special feature). Open 2002-65287, US2002-0025564A, EP1813677A).

  Moreover, in L-glutamic acid fermentation, it can also culture | cultivate, making L-glutamic acid precipitate in a culture medium using the liquid medium adjusted to the conditions which L-glutamic acid precipitates. Examples of conditions under which L-glutamic acid precipitates include pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0. (European Patent Application Publication No. 1078989)

  Recovery of the target substance from the culture solution can usually be carried out by combining ion exchange resin method, precipitation method and other known methods. In addition, when the target substance accumulates in the microbial cells, the target substance is recovered from the supernatant obtained by crushing the microbial bodies with ultrasonic waves and removing the microbial cells by centrifugation, for example, by an ion exchange resin method. can do. When the target substance is an amino acid or a nucleic acid, the target substance to be recovered may be a free form or a salt containing a sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, or potassium salt.

  Moreover, the target substance collected in the present invention may contain microbial cells, medium components, moisture, and microbial metabolic byproducts in addition to the target substance. The purity of the collected target substance is 50% or more, preferably 85% or more, particularly preferably 95% or more (US5,431,933, JP1214636B, US4,956,471, US4,777,051, US4946654, US5,840358, US6, 238,714, US2005 / 0025878).

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

(1) Preparation of piss and ground bagasse Bagasse was obtained from a sugarcane sugar factory. The obtained bagasse was sterilized and dried. Bagasse can be obtained by a sugar refining process from ordinary sugarcane.
The dried bagasse was treated with a 20 mesh (0.85 mm) vibrating sieve to remove the lined, and the fibrous lined was removed from the passed fraction to separate the piss.

The ground bagasse was prepared by the following procedure. The dried bagasse was pulverized with a pulverizer (Nara type automatic pulverizer M-4 type (Nara Machinery Co., Ltd.)) under pulverization conditions: rotational speed 5500 min ⁻¹ , screen 0.9 mm. The pulverized product was sieved and passed through a sieve having a mesh of 0.35 mm, and the one not passing through a 0.15 mm sieve was used as a crushed bagasse. This ground bagasse is obtained by finely cutting a mixture of pith and lined to a size equivalent to that of piss for use as a control of piss.

(2) Saccharification by saccharifying enzyme As a saccharifying enzyme, a commercially available Cellic Ctec enzyme (manufactured by Novozyme) was used. This enzyme contains cellulase derived from Trichoderma. Add 0.15 g of 100 mM acetic acid, 0.0286 g or 0.00286 g of saccharifying enzyme, 0.0025 g of Geneticin (antibiotic), and water so that the solution volume is 10 ml, and add 50 ml to 50 g And shake reaction at 60 rpm for 4 days. Glucose released in the reaction supernatant was measured with a biotech analyzer manufactured by Asahi Kasei. The results are shown in Table 1.
When saccharification of pis, it was possible to recover more glucose than glucose obtained from crushed bagasse with an enzyme amount of 1/10 or less used when saccharified bagasse was saccharified.

Claims (6)

  A method for producing a sugar solution, comprising the step of reacting a pith separated from sugarcane with a saccharifying enzyme in a liquid to produce sugar.   The method of claim 1, wherein the pis is separated from sugarcane bagasse.   The method according to claim 2, wherein the pis is obtained by separating sugar cane bagasse into pis and lined and collecting pis.   The method according to any one of claims 1 to 3, wherein the saccharifying enzyme is cellulase or a mixture of cellulase and hemicellulase.   The manufacturing method of the target substance which manufactures a target substance with a fermentation method by manufacturing a sugar liquid by the method as described in any one of Claims 1-4, and using the obtained sugar liquid or its fraction as a carbon source.   The method according to claim 5, wherein the target substance is an L-amino acid or a nucleic acid.