JP5000189B2 - Method for producing highly efficient organic compound by coryneform bacterium transformant - Google Patents

Description

  The present invention relates to a method for producing an organic compound with high productivity by a microorganism with enhanced ability to carry out a biorefinery process. More specifically, using a coryneform bacterium transformant with enhanced glyceraldehyde 3-phosphate dehydrogenase (hereinafter abbreviated as GAPDH) activity using saccharides derived from biomass as a raw material, improving the metabolic rate of the saccharides The present invention relates to a highly efficient method for producing an organic compound.

  The production of chemical products and energy products such as lactic acid, succinic acid, and ethanol using biomass-derived saccharides as a raw material (biorefinery) differs from the production of these organic compounds from fossil resources (petroleum refinery). It is expected as an environmentally harmonious manufacturing technology that solves the problem of resource depletion and global warming.

However, the production of various organic compounds by biological methods is generally said to be less productive than petrochemical processes.
One method of improving productivity by biological methods is to increase the metabolic rate of saccharides into various organic compounds and increase the concentration of organic compounds in bioreaction solutions (highly efficient separation and purification of target organic compounds). Can be implemented).

  The present inventors have already proposed several techniques relating to a highly productive biological method (see Patent Documents 1, 2, and 3). Patent Document 4 discloses a technique for highly accumulating pyruvate, which is a metabolite from glucose, in a culture solution under aerobic conditions using E. coli with enhanced GAPDH activity, which is one of glycolytic enzymes. Yes.

  GAPDH is an enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, which is one of the glycolytic pathways (see FIG. 4). This metabolic pathway requires a coenzyme, and in this metabolic pathway reaction, the coenzyme nicotinamide dinucleotide (NAD) is reduced to reduced nicotinamide dinucleotide (NADH).

  It is also known that one of the redox coenzyme pairs, NADH, is an inhibitor of GAPDH activity (see Non-Patent Documents 1 and 2). In order to promote metabolic pathway reactions, it is necessary to take measures to remove such factors that cause such reaction inhibition.

  Non-Patent Documents 3 and 4 show that NADH / NAD abundance ratios in microbial cells are different under aerobic conditions (oxidative conditions) and anaerobic conditions (reductive conditions), and NADH / NAD ratios are different under aerobic conditions. Teaching to maintain equilibrium in a low state.

  Patent Document 4 discloses a technique for producing pyruvic acid under aerobic conditions, because the abundance of NADH is lower than that of NAD under aerobic conditions, thereby eliminating the GAPDH inhibitory effect of NADH. As a result, it is considered that the carbonaceous flow from glucose to pyruvic acid is smoothly performed.

  However, the production of organic compounds such as lactic acid, succinic acid and ethanol by biological methods is under anaerobic conditions (reducing conditions) from the viewpoint of selectivity of product yield, ease of reaction control and energy saving of reaction process. It is desirable to implement. There is a need for a technique that can increase the concentration of the organic compound produced in the reaction solution without obstructing the carbonaceous flow in the metabolic pathway even under anaerobic conditions (such as elimination of inhibition of GAPDH activity by NADH). ing.

Patent Document 5 discloses a technique relating to a GAPDH transformant related to a (nicotinamide dinucleotide phosphate / reduced nicotinamide dinucleotide phosphate) (NADP / NADPH) redox coenzyme pair of a eukaryotic microorganism such as yeast. However, it is an enzyme protein that is significantly different from GAPDH related to the (NAD / NADH) redox coenzyme pair of the present invention, and is not related to the transformation technology of coryneform bacteria that are the prokaryotic microorganisms of the present invention. .
WO2005 / 010182 Publication Japanese Patent Application No. 2005-010928 Japanese Patent Application No. 2005-148053 JP 2004-283050 A JP 2005-507255 A C. GARRIGUES, J.A. Bacteriology, Vol. 179, no. 17, 5282-5287 (1997) H. Dominguez, Eur. J. et al. Biochem. 254, 96-102 (1998) M.M. R. Graef, J. et al. Bacteriology, Vol. 181, no. 8, 2351-2357 (1999) C. Du, Appl. Microbiol. Biotechnol. 69: 554-563 (2006)

An object of this invention is to provide the manufacturing method under reducing conditions which can implement useful organic compounds, such as lactic acid, a succinic acid, and ethanol, industrially highly efficient and highly productive.
According to the method of the present invention, it is possible to improve the conversion rate of carbonaceous matter to an organic compound, which is a metabolite of saccharides, and at the same time prevent deterioration of the conversion reaction over time, and improve the accumulated concentration of the organic compound in the bioreaction solution. I can do it.

  As described above, the method for producing a useful organic compound from a saccharide under reducing conditions, which is the object of the present invention, is difficult to eliminate the GAPDH inhibitory effect by NADH in the main metabolic pathway under reducing conditions as described above. Therefore, it was predicted that it would be difficult to implement with high efficiency. However, as a result of intensive studies, it was unexpectedly found that the object of the present invention can be achieved by identifying a microorganism that enhances the expression of GAPDH activity. I found it.

を示す。 That is, the present invention is as follows.
(1) A metabolic rate of saccharides in a reaction solution under reducing conditions using an aerobic coryneform bacterium transformed with a DNA sequence encoding glyceraldehyde 3-phosphate dehydrogenase under an expressible control sequence An organic compound is produced by accumulating an organic compound and recovering the organic compound from the reaction solution.
(2) Any of the aerobic coryneform bacteria to be transformed selected from the group of Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus The method for producing an organic compound as described in (1) above, wherein
(3) Corynebacterium genus Corynebacterium glutamicum R FERM P-18976, ATCC13032, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13746, ATCC13746, ATCC13746, ATCC13746, ATCC13746, ATCC13746 And the method for producing an organic compound according to (2) above, which is any one selected from Corynebacterium efficiens NBRC 1000039.
(4) Brevibacterium genus is Brevibacterium lactofermentum ATCC 13869, Brevibacterium flavum MJ-233 (FERM BP-1497) or MJ-233AB-41 (FER M ) Or any one selected from Brevibacterium ammoniagenes ATCC6872. The method for producing an organic compound according to (2) above, wherein the microorganism is any one selected from Brevibacterium ammoniagenes ATCC6872.
(5) The DNA sequence encoding glyceraldehyde 3-phosphate dehydrogenase is from Corynebacterium glutamicum FERM P-18976, Actinomyces europaeuus ATCC 700353 or Escherichia coli The method for producing an organic compound according to (1) above, wherein the method is derived from ATCC 27325.
(6) The transformed aerobic coryneform bacterium is Corynebacterium glutamicum R / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20875) The manufacturing method of the organic compound as described in said (1).
(7) The transformed aerobic coryneform bacterium is Corynebacterium glutamicum R △ PEPC / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession No. FERM P-20878) The manufacturing method of the organic compound as described in said (1) which is.
(8) The transformed aerobic coryneform bacterium is Corynebacterium glutamicum R ΔPEPC / Actino-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20877) The manufacturing method of the organic compound as described in said (1) which is.
(9) The method for producing an organic compound as described in (1) above, wherein the oxidation-reduction potential of the reaction solution under reducing conditions is -200 millivolts to -500 millivolts.
(10) The organic compound according to (1), wherein the organic compound is at least one selected from monocarboxylic acids, dicarboxylic acids, ketocarboxylic acids, hydroxycarboxylic acids, amino acids, monoalcohols, polyols, and vitamins. Manufacturing method.
(11) Corynebacterium glutamicum R / R-nGAP strain (Incorporated Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession No. FERM P-20875).
(12) Corynebacterium glutamicum R ΔPEPC / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20878).
(13) Corynebacterium glutamicum R ΔPEPC / Actino-nGAP strain (Incorporated Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession No. FERM P-20877).
In addition, ΔPEPC used in the present specification and claims is:

Indicates.

  According to the present invention, it becomes possible to produce a useful organic compound with high productivity and high efficiency by improving the metabolic conversion consumption rate of saccharides, overcoming the low productivity of biological organic compound production technology. Enables refinery construction.

  The aerobic coryneform bacterium transformed by the present invention is not particularly limited as long as it has a saccharide metabolic conversion function. Depending on the type of saccharide and the type of target organic compound, it may be necessary to introduce new functions, such as uptake of saccharides that aerobic coryneform bacteria do not originally have, metabolic pathways, or conversion pathways to target organic compounds. In such a case, these functions may be imparted by a recombination technique or a mutagenesis treatment.

  Examples of such aerobic coryneform bacteria include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus.

  More specifically, Corynebacterium glutamicum R FERM P-18976, ATCC13032, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13655, ATCC13655, ATCC13655, ATCC13755, ATCC14020 or ATCC31831, Corynebacterium efficiens NBRC 1000039, etc. are mentioned.

  Examples of the genus Brevibacterium include Brevibacterium lactofermentum ATCC 13869, Brevibacterium flavum MJ-233 (FERM BP-1497) or MJ-233AB-41 (FER98). Or Brevibacterium ammoniagenes (ATCC6872) etc. are mentioned.

  Examples of the genus Arthrobacter include Arthrobacter Globiformis ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738, and ATCC35698.

  Examples of the genus Mycobacterium include Mycobacterium bovis ATCC 19210 and ATCC 27289.

  Examples of the genus Micrococcus include Micrococcus fredenreichii No. 239 (FERM P-13221), Micrococcus luteus No. 240 (FERM P-13222) or Micrococcus ureae IAM1010.

  Examples of the aerobic coryneform bacterium used in the present invention include Corynebacterium glutamicum R FERM P-18976, Corynebacterium glutamicum ATCC13032, and Brevibacterium lactofermentum B preferable.

  In addition, the aerobic coryneform bacterium transformed by the present invention includes a mutant FERM P that has acquired the cellobiose assimilation ability of a wild-type mutant (eg, Corynebacterium glutamicum R) that exists in nature. -18777, FERM P-18978 strain and the like; see Japanese Patent Application Laid-Open No. 2004-89029), and an artificial strain in which a gene other than the constituent genes of the present invention is recombined (for example, Corynebacterium glutamicum). ) ATCC13032 transformed to have ethanol production ability (FERM BP-7621; see WO 01/96573 A1) and Corynebacterium glutamicum (Corynebac) an artificial strain (FERM P-181979; JP 2004-89029 A) transformed to express the ability to simultaneously assimilate glucose and cellobiose by genetic recombination with the phosphotransferase enzyme II of E. glutamicum R strain ).

  For the purpose of the present invention, these aerobic coryneform bacteria are subjected to a transformation treatment with high expression of GAPDH described in detail below. Alternatively, the GADPH high expression treatment and the order of creating the mutant strain and artificial strain described above may be reversed or simultaneous.

  The DNA fragment containing the GAPDH gene used in the present invention may be possessed by the aerobic coryneform bacterium of the present invention itself or may be derived from a foreign species.

  The gene sequence and enzyme protein characteristics of GAPDH have been extensively investigated in animals, plants, and microorganisms. Regarding microorganisms, the gene sequence and enzyme protein characteristics of GAPDH possessed by the species shown below have already been clarified.

- Escherichia coli (Escherichia coli) (D'Alessio, G., and Josse J.;. Glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, and phosphoglyceromutase of Escherichia coli.Simultaneous purification and physical properties, J Biol Chem, 246 (1971), 4319 -25)

- Corynebacterium glutamicum (Corynebacterium glutamicum) (Eikmanns, B.J;. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase , J Bacteriol., 174 (1992), 6076-86)

- Streptomyces Arena (Streptomyces arenae) (Maurer, K.H., Pfeiffer F., Zehender H., and Mecke D.; Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae, J Bacteriol. , 153 (1983), 930-6)

Aspergillus nidulan (Punt, PJ, Dingmanse MA, Jacobs-Meijsing BJ, Pouwels P. H., and van den Hondal th e cand e s); -3-phosphate dehydrogenase gene of Aspergillus nidulans, Gene., 69 (1988), 49-57)

- Synechococcus sp. (Synechococcus sp) (Sand, O., Petersen I.M., Jorgen J., and Iversen L.;. Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase from Synechococcus sp, Antonie Van Leeuwenhoek, 65 (1994 ), 133-42)

-Bacillus cereus (Suzuki, K., and Imahori K.), Isolation and some properties of biodegradable 3-phosphate dehydrated vegetative vegetative vein.

Bacillus subtilis (Viaene, A., and Dhaese P .; Sequence of the glyceridedehyde-3-phosphate dehydrated sucrose.

  If the base sequence of the DNA fragment containing the GAPDH gene is known, a DNA fragment synthesized according to the sequence can also be used. Even when the DNA sequence is unknown, the enzyme protein can be purified using GAPDH activity as an indicator, and the DNA fragment can be isolated by the usual hybridization technique using the N-terminal amino acid sequence and the partially degraded sequence. It is also possible to obtain fragments by hybridization and PCR methods based on amino acid sequences conserved between GAPDH enzyme proteins. Furthermore, fragments can be obtained by degenerate PCR using mixed primers designed based on other known GAPDH gene sequences.

  As long as the GAPDH gene of the present invention retains GAPDH activity, a part of the base sequence may be substituted with another base, may be deleted, or a new base may be inserted. Furthermore, a part of the base sequence may be rearranged. Any of these derivatives can be used in the present invention. The above-mentioned part may be 1 to several in terms of amino acid residues, for example.

  The DNA fragment containing the GAPDH gene of the present invention is introduced into the aerobic coryneform bacterium described above using a plasmid vector under a regulatory sequence capable of expressing the GAPDH gene. Here, “under the control sequence” means that the GAPDH gene can replicate autonomously by, for example, joint work with a promoter, an inducer, an operator, a ribosome binding site, a transcription terminator, and the like. As a plasmid vector used for such a purpose, any plasmid vector may be used as long as it contains a gene that controls an autonomous replication function in an aerobic coryneform bacterium. Specific examples thereof include, for example, pAM330 (Agric. Biol. Chem., Vol. 48, 2901-2903 (1984) and Nucleic Acids Sym Ser., Vol. 16, 265-267 (1985)) (derived from Brevibacterium lactofermentum 2256). ), PHM1519 (Agric. Biol. Chem., Vol. 48, 2901-2903 (1984)) (derived from Corynebacterium glutamicum ATCC 13058), pCRY30 (App1. Environ. Microbiol., Vol. 57, 759-4) (57, 759-4). pEK0, pEC5, pEKEx1 (Gene, vol. 102, 93-98 (1991)) and p G4 (J.Bacteriol., Vol.159,306-311 (1984)) (Corynebacterium gluatmicum T250 derived) and the like.

  The construction of the plasmid used for the creation of the coryneform bacterium transformant of the present invention is carried out, for example, by using a gene fragment containing a complete GAPDH gene when using a GAPDH gene derived from Corynebacterium glutamicum R strain [corynebacteria Results of whole genome analysis of Corynebacterium glutamicum R strains (Hiroshi Nonaka, Kaori Nakata, Naoko Okai, Mariko Wada, Yumiko Sato, Kos Peter, Masayuki Inui, Hideaki Yukawa, “Cornebacterium Rumika Genomics” April 2003, Yokohama, Agricultural Chemistry Society of Japan 2003 Annual Meeting Abstract, p. 20)) (amplification is described in the examples)] Suitable promoters, after connecting the control sequences terminator, was inserted into suitable restriction enzyme sites of any plasmid vectors that are exemplified above, can be constructed.

  In the above recombinant plasmid, promoters for expressing the GAPDH gene include promoters originally possessed by aerobic coryneform bacteria, but are not limited thereto, and have a function of initiating transcription of the GAPDH gene. Any base sequence may be used. Examples of the terminator under the control sequence arranged downstream of the GAPDH gene include a terminator originally possessed by an aerobic coryneform bacterium, but are not limited thereto. For example, a tryptophan operon derived from E. coli is used. Any base sequence having a function of terminating transcription of the GAPDH gene, such as a terminator, may be used.

As a method for introducing into the aerobic coryneform bacterial plasmid vector containing the GAPDH gene, if the electric pulse method (electroporation method) and CaCl ₂ method or the like method capable GAPDH gene transfer into the aerobic coryneform bacterium It is not particularly limited. Specific examples thereof include, for example, a known method (Agric. Biol. Chem., Vol. 54, 443-447 (1990), Res. Microbiol., Vol. 144, 181-185 (1993)). Can be used.

  As a method for obtaining a coryneform bacterium transformant of the present invention, a drug resistance gene or the like is incorporated into a plasmid vector containing a GAPDH gene according to a conventional method, and the GAPDH gene is introduced onto a plate medium containing the drug at an appropriate concentration. The transformed coryneform bacterium can be selected by applying the treated coryneform bacterium of the present invention. Specific examples of the method include, for example, Agric. Biol. Chem. , Vol. 54, 443-447 (1990), Res. Microbiol. vol. 144, 181-185 (1993).

  As an example of a transformant of aerobic coryneform bacterium created by the above method, specifically, Corynebacterium glutamicum R / R-nGAP strain (National Institute of Advanced Industrial Science and Technology) Patent Biodeposition Center Accession Number FERM P-20875), Corynebacterium glutamicum R △ PEPC / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession Number FERM P-20878), Corynebacterium Corynebacterium glutamicum R △ PEPC / Actino-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) Number FERM P-20877) and the like.

  The aerobic coryneform bacterium thus created in the reaction solution under the specific reducing conditions is obtained from a saccharide as a raw material (in the case of producing a polycarboxylic acid present in the TCA pathway, carbonate ion or the like as a raw material). ), Various organic compounds such as monocarboxylic acids, dicarboxylic acids, ketocarboxylic acids, hydroxycarboxylic acids, amino acids, monoalcohols, polyols, vitamins, etc. It can be produced in the reaction solution with a high accumulated concentration.

  In the method for producing an organic compound according to the present invention, first, the aerobic coryneform bacterium of the present invention is grown and cultured under aerobic conditions.

  The aerobic coryneform bacterium of the present invention can be cultured using a normal nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, and the like. In the culture, for example, glucose or molasses can be used as a carbon source, and as a nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, or the like can be used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate or magnesium sulfate can be used. In addition to these, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid or various vitamins such as biotin or thiamine can be appropriately added to the medium as necessary.

  Culturing can usually be performed at a temperature of about 20 ° C. to about 40 ° C., preferably about 25 ° C. to about 35 ° C. under aerobic conditions such as aeration stirring or shaking. The pH during the culture is in the range of about 5 to 10, preferably about 7 to 8. The pH during the culture can be adjusted by adding an acid or an alkali. The carbon source concentration at the start of the culture is about 1 to 20% (W / V), preferably about 2 to 5% (W / V). The culture period is usually about 1 to 7 days.

  Next, the cultured cells of the aerobic coryneform bacterium of the present invention are collected. The method for recovering and separating the cultured cells from the culture obtained as described above is not particularly limited, and for example, a known method such as centrifugation or membrane separation can be used.

  The collected cultured microbial cells may be treated, and the resulting microbial cell processed product may be used in the next step. The microbial cell treated product may be any cultivated microbial cell that has been subjected to any treatment, and examples thereof include an immobilized microbial cell in which the microbial cell is immobilized with acrylamide or carrageenan.

  Next, the cultured cells of the aerobic coryneform bacterium of the present invention recovered or separated from the culture obtained as described above or the treated cells thereof are used for the reaction of producing organic compounds in the reaction solution under reducing conditions. Provided. The organic compound generation method can be either a batch method or a continuous method.

  In the biochemical reaction under the reducing conditions of the present invention, the growth and division of aerobic coryneform bacteria is completely suppressed, and the conversion rate and conversion efficiency from sugar carbon sources such as glucose to the target organic compound are improved. In addition, substantial complete suppression of secreted by-products accompanying growth can be realized. From this point of view, when the cultured and recovered coryneform bacteria or the treated cells thereof are used in the reaction solution, the environmental conditions during culture inside and outside the transformed cells of the aerobic coryneform bacteria are not brought into the reaction solution. It is recommended to use methods and conditions. That is, it is preferable that the reaction solution is substantially free from a product that is generated during the growth culture process and is present inside and outside the cells. More specifically, secreted by-products that are generated during the growth and culture process and released outside the cells, and substances that are generated by the aerobic metabolic function in the cells and remain in the cells are reacted under reducing conditions. It is recommended that the medium is substantially absent. Such a state is realized, for example, by centrifuging the culture solution after growth culture, membrane separation, etc. and / or leaving the cultured cell bodies for about 2 to 10 hours under reducing conditions.

  In this step, a reaction solution under reducing conditions is used. The reaction solution may have any shape such as solid, semi-solid, or liquid as long as it is under reducing conditions. An essential requirement of the present invention is to cause the biochemical reaction by the metabolic function of the aerobic coryneform bacterium transformant under reducing conditions to produce the desired organic compound.

  The reduction condition in the present invention is defined by the oxidation-reduction potential of the reaction system, and the oxidation-reduction potential of the reaction solution is preferably about -200 mV to -500 mV, more preferably about -250 mV to -500 mV. The reduction state of the reaction solution can be easily estimated to some extent with a resazurin indicator (in the reduction state, decolorization from blue to colorless). However, an oxidation-reduction potentiometer (for example, ORP Electrodes manufactured by BROADELY JAMES) is used accurately. It can be measured by using it. In the present invention, it is preferable to maintain the reducing conditions immediately after adding the microbial cells or processed product to the reaction solution until collecting the organic compound, but at least at the time of collecting the organic compound, the reaction solution is reduced. Any state is acceptable. It is desirable that the reaction solution be kept under reducing conditions for about 50% or more, more preferably about 70% or more, and further preferably about 90% or more of the reaction time. Among them, the oxidation-reduction potential of the reaction solution is maintained at about -200 mV to -500 mV for a time of about 50% or more, more preferably about 70% or more, more preferably about 90% or more of the reaction time. More desirable.

  Specifically, such a reduced state is achieved by the method for preparing cultured cells after the culture, the method for adjusting the reaction medium, the method for maintaining the reducing conditions during the reaction, or the like.

  A known method may be used as a method for adjusting the reaction solution under reducing conditions. For example, a method for preparing an aqueous solution for a reaction medium is, for example, a method for preparing a culture solution for absolute anaerobic microorganisms such as sulfate-reducing microorganisms (Pfenning, N et. Al. (1981): The dissimilarity sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identification of Bacteria, Ed.by Starr, MP et al. P.926-940, Berlin, Springer Verlag. Edition, 1990, 26th edition, published by Sangyo Tosho Co., Ltd.), etc., can be used as a reference to obtain a desired reduced aqueous solution.

  More specifically, the method for adjusting the reaction aqueous solution includes a method of removing the dissolved gas by subjecting the reaction aqueous solution to a heat treatment or a reduced pressure treatment. More specifically, by treating the aqueous reaction solution for about 1 to 60 minutes, preferably about 5 to 40 minutes under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less, The dissolved gas, particularly dissolved oxygen, can be removed to create an aqueous reaction solution under reducing conditions. An appropriate reducing agent (for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.) can be added to prepare a reduced reaction aqueous solution. In some cases, an appropriate combination of these methods also provides a method for preparing an effective aqueous reaction medium solution in a reduced state.

  As a method for maintaining the reduction conditions during the reaction, it is desirable to prevent oxygen contamination from outside the reaction system as much as possible, and a method in which the reaction system is sealed with an inert gas such as nitrogen gas or carbon dioxide gas is usually used. It is done. As a method for more effectively preventing oxygen contamination, in order to efficiently function the metabolic function of the aerobic coryneform bacterium of the present invention during the reaction, addition of a pH maintenance adjustment solution of the reaction system or various nutrients In some cases, it may be necessary to add a solution appropriately. In such a case, it is effective to remove oxygen from the added solution in advance.

  In the organic compound production reaction of the present invention, the reason why the regulation of the redox potential of the production reaction system is effective for the efficient production of the target organic compound is not clear, but the reason for the estimation is described below. . However, the present invention is not limited to the reason for the estimation.

  The organic compound which is the target product of the present invention is a compound produced by a biochemical reaction based on the metabolic function of the aerobic coryneform bacterium of the present invention. Various redox reactions are involved in biochemical reactions in microbial cells, and electrons are transferred. The oxidation-reduction potential is one of the scales showing the difficulty of accepting and donating electrons in the reaction system, but this potential is a variety of reactions (oxidation-reduction reactions) that constitute metabolic pathways occurring in microbial cells. And the state of electronic transfer between the inside and outside of the cell. The redox potential directly measured by the potentiometer is the potential between the reaction solution and the electrode, but the potential of the reaction solution correlates with the reaction occurring in the cell with a certain potential gradient through the cell membrane. That is, the oxidation-reduction potential reflects the sum of oxidation-reduction reactions in the entire reaction system including inside and outside the cell (including the contents and frequency of various reactions).

  Factors affecting the oxidation-reduction potential of the reaction system include various types and concentrations of the reaction system atmosphere gas, reaction temperature, reaction solution pH, and various inorganic and organic substances used to produce the target organic compounds present in the reaction solution. The compound concentration and composition can be considered. The oxidation-reduction potential of the reaction medium in the present invention is shown by integrating the above various influencing factors. Therefore, in the present invention, various chemical reactions are involved in the metabolic pathway to the target organic compound, and these chemical reactions are under the influence of the above factors, but define a reaction state that is a single redox potential. It has been specified as one of the implementation conditions of the invention by finding that the target organic compound is efficiently produced by the scale.

  The reaction liquid contains an organic carbon source (for example, saccharides) that is a raw material for producing an organic compound. Examples of the organic carbon source include substances that can be used for biochemical reactions by the aerobic coryneform bacterium transformant of the present invention.

  Specifically, examples of the saccharide include monosaccharides such as glucose, xylose, arabinose, galactose, fructose or mannose, disaccharides such as cellobiose, sucrose, lactose or maltose, or polysaccharides such as dextrin or soluble starch. It is done. Of these, glucose is preferable.

  The composition of the reaction solution used for the organic compound production reaction includes components necessary for maintaining the metabolic function of the aerobic coryneform bacterium transformant of the present invention or a processed product thereof, that is, carbon sources such as various sugars, It may contain a nitrogen source necessary for protein synthesis, other salts such as phosphorus, potassium or sodium, and a trace metal salt such as iron, manganese or calcium. These addition amounts can be appropriately determined depending on the required reaction time, the type of the target organic compound product, the type of aerobic coryneform bacterium transformant used, and the like. The addition of certain vitamins may be preferred. Further, in relation to the carbon dioxide gas sealing method in the reaction system, carbon dioxide or various carbonates or bicarbonates such as inorganic carbonates may be added to the reaction medium in addition to organic carbon sources such as sugars. Depending on the target organic compound, it may be effective.

  The reaction of the aerobic coryneform bacterium or its cell-treated product with the saccharide is preferably carried out under temperature conditions where the aerobic coryneform bacterium or its cell-treated product can act. It can be appropriately selected depending on the type of the body treatment product.

  Finally, the organic compound produced in the reaction medium as described above is collected. As the method, a known method used in a bioprocess can be used. Examples of such known methods include salting out, recrystallization, organic solvent extraction, esterification distillation separation, chromatographic separation, or electrodialysis, etc. of the organic compound production liquid. The separation, purification and collection method can be determined accordingly.

  Examples of the organic compound that can be produced in the present invention include organic acids, alcohols, amino acids, and vitamins. Examples of organic acids include acetic acid, lactic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, citric acid, cisaconic acid, itaconic acid, isocitric acid, 2-oxoglutaric acid and Examples include alcohols such as ethanol, 1,3-propanediol, glycerol, butanol, 1,4-butanediol, xylitol, and sorbitol. Examples of amino acids include valine and leucine. , Alanine, aspartic acid, lysine, isoleucine or threonine.

  EXAMPLES Hereinafter, although this invention is demonstrated with an Example, this invention is not limited to such an Example. “%” Means “% by weight” unless otherwise specified.

[Example 1] Cloning of GAPDH gene
(A) Extraction of total DNA from Corynebacterium glutamicum R strain A medium 1 L [composition: urea: 2 g, (NH ₄ ) ₂ SO ₄ : 7 g, KH ₂ PO ₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO ₄ · 7H ₂ O: 0.5 g, FeSO ₄ · 7H ₂ O: 6 mg, MnSO ₄ · nH ₂ O: 4.2 mg, D-biotin: 200 µg, thiamine hydrochloride: 200 µg, yeast 2 g of extract, 7 g of casamino acid, 20 g of glucose, and distilled water: 1000 ml (pH 6.6)] were inoculated with wild strain Corynebacterium glutamicum R using platinum ears until the late logarithmic growth phase. The cells were cultured at 0 ° C. and the cells were collected.

  15 ml of a solution containing each component of 10 mg / ml lysozyme, 10 mM NaCl, 20 mM Tris buffer (pH 8.0) and 1 mM EDTA · 2Na so that the obtained bacterial body has a concentration of 10 mg / ml (concentration of each component) Is the final concentration). Next, proteinase K was added to a final concentration of 100 μg / ml and incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate (SDS) was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. After adding an equal amount of phenol / chloroform solution to this lysate and shaking gently at room temperature for 10 minutes, the whole amount was centrifuged (5,000 × g, 20 minutes, 10 to 12 ° C.) to obtain a supernatant fraction. The fraction was collected. After adding sodium acetate to this supernatant to 0.3M, 2 times amount of ethanol was slowly added. DNA existing between the aqueous layer and the ethanol layer was scraped off with a glass rod, washed with 70% ethanol, and then air-dried. To the obtained DNA, 5 ml of a 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and allowed to stand at 4 ° C. overnight, and used for the subsequent experiments.

(B) Construction of Coryneform Bacteria-Escherichia coli Shuttle Vector pCRB1 Plasmid pAM330 (Yamaguchi, R. et al., Agric. Biol. Chem. 50, 2771-78) endogenous to Brevibacterium lactofermentum ATCC 13869. 1986), and Japanese Laid-Open Patent Application No. 58-67679), a DNA fragment containing ORF1 (rep) was amplified by the following PCR method.

In the PCR, the following pair of primers were synthesized and used using “394 DNA / RNA synthesizer” manufactured by Applied Biosystems.
ORF1 (rep) gene amplification primer Rep-N; 5′-CTCT GTTAAC ACAACAAGACCCATCATAGT-3 ′ (SEQ ID NO: 1),
Rep-C; 5′-CTCT GTTAAC ACATGCAGTCATGTCGTGCT-3 ′ (SEQ ID NO: 2)
Each primer has an HpaI site added to the end.
As the template DNA, plasmid pAM330 was used.
PCR was performed under the following conditions using “DNA thermal cycler” manufactured by Perkin Elmer Citas Co., Ltd. and using Takara Ex Taq (Takara Razo Co., Ltd.) as a reaction reagent.

Reaction solution:
(10 ×) PCR buffer 10 μl
1.25 mM dNTP mixture 16 μl
Template DNA 10 μl (DNA content 1 μM or less)
1 μl of each of the above two primers (final concentration 0.25 μM)
Takara EX Tuck DNA Polymerase 0.5μl
Sterile distilled water 61.5 μl
The above was mixed and 100 μl of the reaction solution was subjected to PCR.

PCR cycle:
Denaturation process: 94 ° C., 60 seconds Annealing process: 52 ° C., 60 seconds Extension process: 72 ° C. 120 seconds or more was defined as one cycle, and 30 cycles were performed.
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 1.8 kb containing the Rep gene could be detected.

  On the other hand, when constructing a coryneform bacterium-E. Coli shuttle vector, the EcoRV was newly added by PCR in order to preserve the lacZα gene of E. coli vector pHSG398 (Takara Shuzo) containing the chloramphenicol resistance gene and the multicloning site therein. PHSG398 was amplified to add sites.

In the PCR, the following pair of primers were synthesized and used using “394 DNA / RNA synthesizer” manufactured by Applied Biosystems.
Primer for amplification of plasmid pHSG398 398-N; 5′-CTCT GATATC GTTCCACTGAGCGTCAGACC-3 ′ (SEQ ID NO: 3),
398-C; 5′-CTCT GATATC TCCGTCGAACGGAAGATCAC-3 ′ (SEQ ID NO: 4)
Each primer has an EcoRV site added to the end.
As the template DNA, plasmid pHSG398 was used.
PCR was performed under the same conditions as described above.
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 2.2 kb including the full length of plasmid pHSG398 could be detected.

  Next, 5 μl of an about 1.8 kb DNA fragment containing the Rep gene cut with HpaI and an about 2.2 kb DNA fragment containing the entire length of plasmid pHSG398 cut with EcoRV were mixed, and this was mixed with 10 × buffer of T4 DNA ligase. Each component of 1 μl of liquid and 1 unit of T4 DNA ligase was added, made 10 μl with sterilized distilled water, reacted at 15 ° C. for 3 hours, and bound.

  Using the obtained plasmid mixture, Escherichia coli JM109 (manufactured by Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg of chloramphenicol [10 g tryptone] , 5 g of yeast extract, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].

The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with a restriction enzyme to confirm the inserted fragment. As a result, in addition to the DNA fragment of plasmid pHSG398 2.2 kb, an inserted DNA fragment having a length of about 1.8 kb was observed.
This coryneform bacteria-E. Coli shuttle vector is referred to as pCRB1.

(C) Cloning of GAPDH gene of Corynebacterium glutamicum R In order to clone GAPDH gene during PCR, the results of whole genome analysis of Corynebacterium glutamicum R strain (Hirori Nonaka, Nakanaka) , Naoko Okai, Mariko Wada, Yumiko Sato, Kos Peter, Masayuki Inui, Hideaki Yukawa “Corynebacterium glutamicum R Genome Analysis”, Japanese Agricultural Chemistry Society, April 2003, Yokohama, Japan Agricultural Chemistry Society Annual Meeting 2003, p. 20)), the following pair of primers was applied to “394 DNA / R” manufactured by Applied Biosystems. Synthesized using the A synthesizer (synthesizer) ", it was used.
GAPDH gene amplification primer GAPR-N; 5′-CTCT GGATCC CGATTTCAGGTTCGTTCCCT-3 ′ (SEQ ID NO: 5),
GAPR-C; 5′-CTCT GGATCC CCGGGCTATTGGGATGA-3 ′ (SEQ ID NO: 6)
Each primer has a BamHI site added to the end.
As the template DNA, the genomic DNA of the Corynebacterium glutamicum R strain extracted in the above section (A) was used.

PCR was performed under the same conditions as in section (B) above.
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and in the case of the GAPDH gene, a DNA fragment of about 1.4 kb could be detected.

  Next, 10 μl of the 1.4 kb PCR product containing the GAPDH gene was cleaved with the restriction enzyme BamHI, and 2 μl of the coryneform bacteria-E. Coli shuttle vector pCRB1 constructed in the above (B) was cleaved with the restriction enzyme BamHI. After inactivating the restriction enzyme by treating at 70 ° C. for 10 minutes, both were mixed, and each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added thereto, and 10 μl with sterile distilled water. And reacted at 15 ° C. for 3 hours. Using this ligation solution, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and chloramphenicol 50 mg, x-gal (5- Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (200 mg), IPTG (isopropyl 1-thio-beta-d-galactoside) 100 mg (trypton 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g) Dissolved in 1 L of water].

A growing strain showing a white color on the medium was selected and subjected to liquid culture by a conventional method. Plasmid DNA was extracted from the culture, cut with a restriction enzyme, and the inserted fragment was confirmed. As a result, in addition to the DNA fragment of plasmid pCRB1 of about 4.0 kb, an inserted DNA fragment of about 1.4 kb in length containing the GAPDH gene (SEQ ID NO: 7) was observed (SEQ ID NO: 7).
The plasmid containing the GAPDH gene was named pCRB200 (see FIG. 1).

(D) Cloning of the GAPDH gene of Actinomyces europaeus (ATCC 7000035) Total DNA from Actinomyces europaeus was extracted as in (A).
The following primers were synthesized using the homologous region of the gapA amino acid sequence of the G (+) bacterium as the same as A. europaeus as an index, and the total DNA purified from A. europaeus was used as a template. Then, PCR was performed using TaKaRa LA Taq (manufactured by Takara Shuzo Co., Ltd.) under the same conditions as in section (B) above.
Actinomyces europaeus (A. europaeus) GAPDH gene amplification primer Agp P1 5'-ATIAAYGGITTYGGIMGIATIGG-3 '(SEQ ID NO: 8),
Agp P7 5′-SCIYAYTCRTTRTCRTA-3 ′ (SEQ ID NO: 9)
Electrophoresis was performed on a 0.8% agarose gel, and a band of about 900 bp was obtained.
The above amplified DNA fragment mixture was purified under the same conditions as in the above section (B), and then reacted at 16 ° C. for 3 hours according to the company's manual using pGEMT-easy Vector system I (Promega). Combined.

  Using the obtained plasmid mixture, Escherichia coli JM109 (Takara Shuzo Co., Ltd.) was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970), and a medium containing 50 mg of ampicillin (10 g tryptone, yeast 5 g of extract, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].

  The growing strain on this medium was subjected to liquid culture by a conventional method, and a plasmid DNA into which a 936 bp PCR product had been inserted was extracted from the culture solution.

Using the plasmid as a template, the following two primers were used to label with Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, USA), and ABI Prism 3100 Genetic Analyzer (Applied Base, USA) The sequence was determined. The sequence determined by the primer showed 60% or more homology with the amino acid sequence of the closely related GAPDH registered in the database.
M13-M4 5′-GTTTTCCCAGTCACGAC-3 ′ (SEQ ID NO: 10),
M13-RV 5′-CAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 11)
As a template for Southern hybridization, seven types of restriction enzyme restriction enzymes (AccI, SphI, BamHI, EcoRI, PstI, SmaI, SalI) 5 U are used to fragment the above-mentioned genome of A. europaeus (A. europaeus). And purified according to the method of (A).

A partial base sequence (936 bp) of GAPDH derived from A. europaeus determined by the above sequence was purified according to the method of item (A) to obtain a probe for genomic Southern.
DNA fragments each fragmented with 6 types of restriction enzymes were electrophoresed, and then hybridized with the probe. Complex formation between the probe and template DNA was identified by fluorescence detection using alkaline phosphatase.
As a result, about 3-kb and 5.5-kb bands were detected in the DNA samples treated with AccI and SphI.

The Accinomyces europaeus-derived DNA treated with AccI and PstI was self-ligated using T4 DNA ligase according to the method of (B).
The formed circular DNA was purified according to the method of (B) and used as a PCR template. Based on the partial base sequence (936 bp) of GAPDH derived from A. europaeus determined so far, the primers shown below were designed.
Contig-R 5′-CGCACTCGTCGATCTTCGAC-3 (SEQ ID NO: 12)
Contig-F 5′-TGATGGACTCGTCATCGTAG-3 ′ (SEQ ID NO: 13)
PCR was performed according to the method of (B). As a result of electrophoresis according to the method of (B), a band of about 4.5-kb was obtained from the genomic sample cut with SphI.
The obtained band was purified according to the method of (B) and cloned using the above-described pGEMT-easy Vector system I (Promega).
Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed according to the method of (B) using a plasmid solution into which an about 4.5-kb PCR product had been inserted, and a medium containing 50 mg of ampicillin [trypton 10 g, yeast 5 g of extract, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].
The growing strain on this medium was subjected to liquid culture by a conventional method, and plasmid DNA was extracted and purified from the culture solution according to the method described in (B).

By using the plasmid as a template and sequencing according to the above method, a base sequence of 1296 bp including the full-length GAPDH derived from A. europaeus was obtained, and the entire base sequence of GAPDH was determined.
The following primers were synthesized based on the above 1296 bp base sequence, and PCR was performed using the genome of A. europaeus as a template.
genm-Eco1;
5′-CTCTGAATTCTAAATCTATCGAAACGGTGT-3 ′ (SEQ ID NO: 14),
genm-Pst5;
5′-CTCTGACGTCTCTTCCTCTCGAAGTTCAAT-3 ′ (SEQ ID NO: 15)
As a result of electrophoresis of the PCR product according to the method of (B), a band of 1240-bp was obtained.

  10 μl of 1.2 kb PCR product containing the GAPDH gene cut with restriction enzymes EcoRI and PstI, and 2 μl of the coryneform bacterium-E. Coli shuttle vector pCRB1 constructed in the above section (B) cut with restriction enzymes EcoRI and PstI After inactivating each of the restriction enzymes by treating at 70 ° C. for 10 minutes, both were mixed, and each of the components of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added thereto, and then sterilized with distilled water. 10 μl was allowed to react at 15 ° C. for 3 hours. Using this ligation solution, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 50 mg of chloramphenicol [trypton 10 g, 5 g of yeast extract, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].

  A growing strain showing a white color on the medium was selected and subjected to liquid culture by a conventional method. Plasmid DNA was extracted from the culture, cut with a restriction enzyme, and the inserted fragment was confirmed. As a result, in addition to a DNA fragment of about 4.0 kb derived from plasmid pCRB1, an inserted DNA fragment of about 1.8 kb in length derived from the GAPDH gene was confirmed (SEQ ID NO: 16).

  Hereinafter, a plasmid containing the GAPDH gene derived from Actinomyces europaeuus ATCC 700353 can be obtained by the same method as in (B), and this plasmid was named pCRA820 (see FIG. 2).

[Comparative Example 1]
Creation of PEPC gene disruption strain by markerless gene disruption method (A) Construction of markerless gene disruption vector pCRA725 A DNA fragment containing the sacR-sacB gene of Bacillus subtilis strain was amplified by the following PCR method .
In the PCR, the following pair of primers were synthesized and used using “394 DNA / RNA synthesizer” manufactured by Applied Biosystems.
sacR-sacB gene amplification primer sacR-sacB-N;
5′-CTCT CAATTG ATAAAGCAGGCAAGACCTAA-3 ′ (SEQ ID NO: 17),
sacR-sacB-C;
5′-CTCT GATATC TTTATTTGTTAACTGTTAAT-3 ′ (SEQ ID NO: 18)
The former has a MunI site and the latter has an EcoRV site added to the end.

As the template DNA, plasmid pMV5 (Vertes, A. A. et al. Isolation and charactersat on of IS 31831, a transposable element from Corynebacterium glutamicum. Mol. Microbiol. 11, 19-94) was used.
PCR was performed under the same conditions as in Example 1 (B).
10 μl of the resulting reaction solution was electrophoresed on a 0.8% agarose gel to detect an approximately 1.6 kb DNA fragment containing the sacR-sacB gene.

  Next, 10 μl of a 1.6 kb PCR product containing the sacR-sacB gene cleaved with MunI and EcoRV and 2 μl of an about 4.6 kb expression vector pKK223-3 (Pharmacia) cleaved with EcoRI and SmaI, respectively. After inactivating the restriction enzyme by treating at 70 ° C. for 10 minutes, both were mixed, and each component of T4 DNA ligase 10 × buffer and 1 μl of T4 DNA ligase and 1 unit of T4 DNA ligase were added to 10 μl with sterile distilled water. And reacted at 15 ° C. for 3 hours. Using this ligation solution, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 10 mg of ampicillin (10 g tryptone, yeast extract). 5 g, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].

The growing strain on this medium was subjected to liquid culture by a conventional method, and plasmid DNA was extracted from the culture solution to obtain vector pCRA724.
Next, sacR-sacB gene (Ptac-sacR-sacB) added with tac promoter was amplified using pCRA724 as template DNA.

In the PCR, the following pair of primers were synthesized and used using “394 DNA / RNA synthesizer” manufactured by Applied Biosystems.
Ptac-sacR-sacB amplification primer Ptac-sacR-sacB-N;
5′-CTCT GATATC CATAATTCGTGTCGCTCAAG-3 ′ (SEQ ID NO: 19)
sacR-SacB-N;
5′-CTCT GATATC TTTATTTGTTAACTGTTAAT-3 ′ (SEQ ID NO: 20),
Each primer has an EcoRV site added to the end.
PCR was performed under the same conditions as in Example 1 (B).
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 1.8 kb containing the Ptac-sacR-SacB gene was detected.

  Next, 5 μl of an approximately 1.8 kb DNA fragment containing the above Ptac-sacR-SacB gene digested with EcoRV and 2 μl of approximately 2.7 kb plasmid pHSG298 (manufactured by Takara Shuzo Co., Ltd.) digested with StuI were each prepared at 70 ° C. After inactivating the restriction enzyme by treating for 10 minutes, both were mixed, and each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added thereto, and the volume was made 10 μl with sterile distilled water. , Reacted at 15 ° C. for 3 hours to bind.

Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 10 mg of kanamycin (10 g of tryptone, yeast extra). 5 g of NaCl, 5 g of NaCl, and 16 g of agar were dissolved in 1 L of distilled water].
The growth strain on the above medium was subjected to liquid culture by a conventional method, and plasmid DNA was extracted from the culture solution to obtain a markerless gene disruption vector pCRA725.

(B) Cloning of PEPC gene and creation of plasmid for gene disruption PCR was performed using the chromosomal DNA prepared in Example 1 (A) as a template.
In the PCR, in order to clone the PEPC gene, results of whole genome analysis of Corynebacterium glutamicum R strain (Hiroshi Nonaka, Kaori Nakata, Naoko Okai, Mariko Wada, Yumiko Sato, Kos Peter, Masayuki Inui, Based on Hideaki Yukawa, “Corynebacterium glutamicum R Genome Analysis”, Japanese Society of Agricultural Chemistry, April 2003, Yokohama, Japan Agricultural Chemical Society 2003 Annual Meeting Abstracts, p. 20), the following pair of primers were applied: -It synthesized and used using "394 DNA / RNA synthesizer" (Applied Biosystems) company.
PEPC gene amplification primer PEPC-N; 5′-CTCT GTCGAC ATGACTGATTTTCTACGCGA-3 ′ (SEQ ID NO: 21),
PEPC-C; 5′-CTCT GCATGC CTAGCCGGAGTTGCGCAGTG-3 ′ (SEQ ID NO: 22)
The former is added with a SalI site, and the latter is added with a SphI site.

As the template DNA, the genomic DNA of Corynebacterium glutamicum R strain extracted in Example I (A) was used.
PCR was performed under the same conditions as in Example 1 (B).
10 μl of the reaction solution generated above was electrophoresed on a 0.8% agarose gel, and a DNA fragment of about 2.8 kb containing the PEPC gene was detected.

  Next, 10 μl of a DNA fragment of about 2.8 kb containing the PEPC gene cleaved with the restriction enzymes SalI and SphI, and a markerless gene disruption of about 4.4 kb constructed in the above section (A) cleaved with SalI and SphI After inactivating the restriction enzyme by treating each 2 μl of vector pCRA725 at 70 ° C. for 10 minutes, both were mixed, and each component of T4 DNA ligase 10 × buffer 1 μl and T4 DNA ligase 1 unit was added and sterilized. It was made 10 μl with distilled water and reacted at 15 ° C. for 3 hours. Using this ligation solution, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and kanamycin 50 mg, x-gal (5-Bromo-4 -Chloro-3-indoxyl-beta-D-galactopylanoside (200 mg), IPTG (isopropyl 1-thio-beta-d-galactoside) 100 mg medium (tryptone 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g in distilled water 1 L Smeared.

A growing strain showing a white color on the medium was selected and subjected to liquid culture by a conventional method. After extracting plasmid DNA from the culture solution, it was cleaved with a restriction enzyme to confirm the inserted fragment. As a result, in addition to the DNA fragment of plasmid pCRA725 approximately 4.4 kb, an inserted DNA fragment of about 2.8 kb in length containing the PEPC gene was observed.
The plasmid containing the PEPC gene was designated as pCRA725-PEPC.

  Next, 2 μl of PECR gene markerless disruption vector pCRA725-PEPC cleaved with XhoI was inactivated at 70 ° C. for 10 minutes, and then the restriction enzyme was inactivated. Then, T4 DNA ligase 10 × buffer 1 μl, T4 DNA ligase 1 unit Were added to 10 μl with sterilized distilled water and reacted at 15 ° C. for 3 hours. Using this ligation solution, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed by the calcium chloride method [Journal of Molecular Biology, 53, 159 (1970)], and a medium containing 10 mg of kanamycin [tryptone 10 g, yeast extract]. 5 g, 5 g of NaCl and 16 g of agar were dissolved in 1 L of distilled water].

Growing strains were selected on the above medium and subjected to liquid culture by a conventional method. Plasmid DNA was extracted from the culture and then cleaved with a restriction enzyme to confirm the deletion of about 1.1 kb in the central part of the PEPC gene. As a result, in addition to the DNA fragment of plasmid pCRA725 about 4.4 kb, a DNA fragment of about 1.7 kb in length containing the ΔPEPC gene fragment was observed.
The PEPC gene markerless disrupting plasmid was named pCRA725-ΔPEPC.

(C) Creation of PEPC Gene Disrupted Strain PEPC gene markerless disrupting plasmid pCRA725-ΔPEPC is a plasmid that cannot replicate in the genus Corynebacterium (including Corynebacterium glutamicum R strain). pCRA725-ΔPEPC was prepared using the electric pulse method (Y. Kurusu, et al., Agric. Biol. Chem. 54: 443-447.1990. and AA Vertes, et al., Res. Microbiol. 144: 181. -A. Agar medium (1 L [composition: urea: 2 g, (NH ₄ ) ₂ SO ₄ : 7 g) introduced into Corynebacterium glutamicum R strain according to the method of 185.1993) and containing 50 μg / ml of kanamycin. , KH ₂ PO ₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO ₄ · 7H ₂ O: 0.5 g, FeSO ₄ · 7H ₂ O: 6 mg, MnSO ₄ · nH ₂ O: 4.2 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast Scan 2g, was applied casamino acid 7 g, glucose 20g, to 1000ml dissolved in distilled water agar 16g (pH6.6)].

Further, the strain obtained in the above medium was added to a sucrose-containing minimal medium (1 L [composition: urea: 2 g, (NH ₄ ) ₂ SO ₄ : 7 g, KH ₂ PO ₄ : 0.5 g, K ₂ HPO ₄ : 0 0.5 g, MgSO ₄ .7H ₂ O: 0.5 g, FeSO ₄ .7H ₂ O: 6 mg, MnSO ₄ .nH ₂ O: 4.2 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, sucrose: 100 g, agar 16 g was dissolved in 1000 ml of distilled water].

  When plasmid pCRA725-ΔPEPC undergoes one-point homologous recombination with a wild-type gene on the chromosome, kanamycin resistance due to expression of kanamycin resistance gene on vector pCRA725 and sucrose lethality due to expression of sacR-sacB gene are shown On the other hand, when two-point homologous recombination occurred, kanamycin sensitivity due to loss of the kanamycin resistance gene on the vector pCRA725 and growth in a sucrose-containing medium due to loss of the sacR-sacB gene are shown. Therefore, the target PEPC gene-disrupted strain exhibits kanamycin sensitivity and sucrose-containing medium growth.

A strain showing kanamycin sensitivity and sucrose-containing medium growth was isolated, liquid-cultured by a conventional method, chromosomal DNA was extracted from the recovered cells by the same method as in Example 1 (A), and sequencer Prism 3100 genetic analyzer. (ABI) confirmed the destruction of the PEPC gene on the chromosome. The PEPC gene disrupted strain thus obtained was named Corynebacterium glutamicum R / ΔPEPC strain (Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession Number; FERM P-2087).
The disappearance of the PEPC activity in the Corynebacterium glutamicum R / ΔPEPC strain was confirmed by the following method.

Corynebacterium glutamicum R / ΔPEPC strain was added to A medium 100 ml (1 L [composition: urea: 2 g, (NH ₄ ) ₂ SO ₄ : 7 g, KH ₂ PO ₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO ₄ · 7H ₂ O: 0.5 g, FeSO ₄ · 7H ₂ O: 6 mg, MnSO ₄ · nH ₂ O: 4.2 mg, D-biotin: 200 µg, thiamine hydrochloride: 200 µg, yeast extract 2 g , Casamino acid 7 g, glucose 20 g and distilled water: 1000 ml (pH 6.6)]) using a platinum loop and culturing at 33 ° C. until the late logarithmic growth phase to collect the cells. The cells Tris buffer _{(100mM Tris-HCl (pH7.5)} , 20mM KCl, 20mM MgCl 2, 5mM MnSO 4, 0.1mM EDTA, 2mM DTT) were washed once with. 0.5 g of this washed microbial cell was suspended in 2 ml of the same buffer, and a microbial cell lysate was obtained using an ultrasonic crusher (Astrason model XL2020) under ice cooling. The crushed material was centrifuged (10,000 × g, 4 ° C., 30 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of the wild type Corynebacterium glutamicum R strain was similarly prepared and subjected to the following activity measurement. The activity of PEPC was measured by measuring the amount of coenzyme NADH oxidized to NAD ⁺ with malic acid production using phosphoenolpyruvate as a substrate (Jetten, M., and Sinkey A). J .; Characteristic of phosphoenolpyrate carboxykinase from Corynebacterium glutamicum, FEMS Microbiology Letters, 111 (1993), 183-188.). As a result, PEPC activity was not detected in Corynebacterium glutamicum R / ΔPEPC strain, confirming the disruption of the PEPC gene.

[Example 2] GAPDH activity-enhanced strain creation of Corynebacterium glutamicum R (National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession Number: FERM P-18976) Kurusu, et al., Agric.Biol.Chem.54: 443-447.1990. And AA Vertes, et al., Res.Microbiol.144: 181-185.1993). It was introduced into the Corynebacterium glutamicum R strain.

Recombinant cell name; Corynebacterium glutamicum R / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession Number; FERM P-20875)
In addition, the GAPDH activity of Corynebacterium glutamicum R / R-nGAP strain is determined by the GAPDH activity measurement method (Okumasaba, CA, Okai N., Inui M., and Yukawadehc H .; 3-phosphate dehydrogenase with opposite, ATP-dependent regulation, J Mol Microbiol Biotechnol., 8 (2004), 91-103, compared to the wild strain (Corynebacterium glutamicum 3) Double life Increase was observed.

Similarly, the plasmid pCRB200 was introduced into the Corynebacterium glutamicum R / ΔPEPC strain by the above method.
Recombinant cell name; Corynebacterium glutamicum R / ΔPEPC / R-nGAP strain (Incorporated Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession Number; FERM P-20878)
In addition, the GAPDH activity of Corynebacterium glutamicum R.DELTA.PEPC / R-nGAP strain was measured by the GAPDH activity measurement method (Okumasaba, CA, Okai N., Inui M., and Yukawaum. Glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation, J Mol Microbiol Biotechnol., 8 (2004), 91-103, A 5.4-fold increase in activity was observed.

Similarly, the plasmid pCRA820 was introduced into the Corynebacterium glutamicum R / ΔPEPC strain by the above method.
Recombinant cell name; Corynebacterium glutamicum R / ΔPEPC / Actino-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession Number; FERM P-20877)
The GAPDH activity of the recombinant cell name; Corynebacterium glutamicum R / ΔPEPC / Actino-nGAP strain was determined by the GAPDH activity measurement method (Okumasaba, CA, Okai N., Inui M., and Yukawa H .; Corynebacterium glutamicum glycerdehyde-3-phosphate dehydrum with hydroid, Bacterial strain, ATP-dependent regulation, JMol. cum) R / △ PEPC)) 5.6-fold increased activity as compared to was observed.

[Example 3] Aerobic culture growth and lactic acid production reaction experiment of recombinant strain (A) Aerobic culture growth Corynebacterium glutamicum R / R-nGAP strain stored in a freezer at -80 ° C (Created in Example 2), A agar medium (composition: urea 2g, yeast extract 2g, casamino acid 7g, ammonium sulfate 7g, monobasic potassium phosphate (KH ₂ PO ₄ ) 0.5g) Dibasic potassium phosphate (K ₂ HPO ₄ ) 5 g, magnesium sulfate heptahydrate 0.5 g, iron sulfate heptahydrate 6 mg, manganese sulfate monohydrate 4.2 mg, biotin 0.2 mg, thiamine 0.2 mg, glucose 40 g, agar 1.5% (W / V) and distilled water 1 L), and allowed to stand in a dark place at 33 ° C. for 12 hours.

  Corynebacterium glutamicum R / R-nGAP strain grown on the above plate was mixed with the above-mentioned A agar medium except that A medium (composition: no agar was included) (Ingredient composition) 10 ml was inoculated with platinum ears and cultured with shaking at 33 ° C., 12 hr, 200 rpm.

  The coryneform bacterial strain thus grown contains 500 ml of an AU medium (composition: the same composition as that of the A medium except that it does not contain urea), which is an aerobic culture growth medium. It was transferred to a 1 L jar fermenter, and aerobic culture growth was carried out for 13 hours by aeration at 33 ° C., 1000 rpm, and sterilized air at 1 vvm.

During this time, NH ₄ OH (5N aqueous ammonia solution) was used to constantly maintain the pH in the jar fermenter tank at 7.5.
The cells grown in this manner were collected by centrifugation (4 ° C., 10 minutes, 5000 × G) and subjected to the lactic acid production reaction under the following reducing conditions.

(B) Lactic acid production reaction under reducing conditions 150 g of wet cells recovered from the aerobic culture growth step in (A) (about 30 g in terms of Dry Cell) were used as a BT-U medium (composition) as a solution for succinic acid production reaction. : Ammonium sulfate 7 g, primary potassium phosphate (KH ₂ PO ₄ ) 0.5 g, dibasic potassium phosphate (K ₂ HPO ₄ ) 0.5 g, magnesium sulfate heptahydrate 0.5 g, iron sulfate 7 water hydrate 6 mg, manganese monohydrate 4.2mg sulfate, biotin 0.2 mg, thiamin 0.2 mg, glucose 36 g, sodium bicarbonate _(NaHCO 3) 12.6 g, reaction of distilled water 1L) capacity containing a 500 ml 1L In addition to the tank, the mixture was gently stirred at 33 ° C., and a succinic acid production reaction under reducing conditions was carried out for 48 hours. The oxidation-reduction potential of the medium during the reaction decreased rapidly immediately after the start of the reaction, and thereafter maintained at about −400 mV, and the succinic acid production reaction was continued.

During this time, the pH in the reaction vessel was constantly maintained at 7.5 using an aqueous NH ₄ OH (5N aqueous ammonia) solution having a concentration of 5N (normal).
The glucose concentration in the reaction vessel was successively sampled and analyzed with a glucose sensor (manufactured by Oji Scientific Instruments Co., Ltd.) to examine the fluctuation of the production amount over time.
The amount of lactic acid produced in the reaction vessel was successively sampled and analyzed by liquid chromatography.
The results are shown in FIG. (The glucose consumption rate and the lactic acid production rate in FIG. 5 are the consumption and production per unit time at 24 hours and 48 hours after the start of the reaction.)
The glucose consumption rates of the Corynebacterium glutamicum R / R-nGAP strains after 24 hours and 48 hours were 1.58 g / l / h and 1.37 g / l / h, respectively.
The production rates of lactic acid were 2.07 g / l / h and 0.97 g / l / h, respectively.
These results are shown in FIG.

[Comparative Example 2]
It was aerobic under the same conditions as in Example 3 except that Corynebacterium glutamicum R strain was used instead of Corynebacterium glutamicum R / R-nGAP strain used in Example 3. Culture growth and lactic acid production reaction were performed.
The consumption rate of glucose of Corynebacterium glutamicum R strain after 24 hours and 48 hours was 1.44 g / l / h and 1.24 g / l / h, respectively.
The production rates of lactic acid were 0.81 g / l / h and 0.36 g / l / h, respectively.
These results are shown in FIG.
The experimental results of Example 3 and Comparative Example 2 indicate that the glucose consumption rate and the organic compound (lactic acid) production rate are increased by high expression of GAPDH in Glutamicum (Corynebacterium glutamicum) R strain.

Example 4
Corynebacterium glutamicum R / ΔPEPC strain (created in Comparative Example 1) and Corynebacterium glutamicum turium camicum (Corynebacterium glutamicum) were used instead of Corynebacterium glutamicum R strain used in Comparative Example 2, respectively. ) Example 3 with the exception of using the R / ΔPEPC / R-nGAP strain (created in Example 2), Corynebacterium glutamicum R / ΔPEPC / Actino-nGAP strain (created in Example 2). The aerobic culture growth and lactic acid production reaction were performed under the same conditions. The results are shown in FIG.

  The Corynebacterium glutamicum R / ΔPEPC strain is less selective in terms of glucose consumption than the wild-type Corynebacterium glutamicum R, but has a higher selectivity for lactic acid. Is known to be a strain capable of producing (Inui, M., Murakami S., Okino S., Kawaguchi H., Vertes A., and Yukawa H.; Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen-deprivation conditions, J.Mol.Microbiol.Biotechnol , Accepted (2004))).

  As is apparent from FIG. 6, the glucose consumption rates of Corynebacterium glutamicum R / ΔPEPC strains after 24 hours and 48 hours were 1.08 g / l / h and 1.06 g, respectively. The glucose consumption rates of the Corynebacterium glutamicum R / ΔPEPC / R-nGAP strains were 1.98 g / l / h and 1.96 g / l / h, respectively. there were. The glucose consumption rates of Corynebacterium glutamicum R / ΔPEPC / Actino-nGAP strains are 2.12 g / l / h and 1.87 g / l / h, respectively.

  Regarding the lactic acid production rate, the production rates of lactic acid after 24 hours and 48 hours of Corynebacterium glutamicum R / ΔPEPC strain were 0.15 g / l / h and 0.15 g / l, respectively. However, the production rates of lactic acid of Corynebacterium glutamicum R / ΔPEPC / R-nGAP strains were 0.99 g / l / h and 0.91 g / l / h, respectively. It was. Moreover, the production | generation rates of lactic acid of Corynebacterium glutamicum R / (DELTA) PEPC / Actino-nGAP strain | stump | stock are 0.91 g / l / h and 1.03 g / l / h, respectively.

  As for these experimental results, as for the Corynebacterium glutamicum R / ΔPEPC strain, as with the wild strain, due to the high expression of GAPDH (heterologous or of its own origin), the consumption rate of glucose and substances It shows that the production speed increases. And even if it is an artificial mutant of an aerobic coryneform bacterium other than about GAPDH, it is clear that the effect of this invention is produced.

  By using the method for producing an organic compound according to the present invention, it is possible to improve the metabolic conversion consumption rate of saccharides and produce a useful organic compound with high productivity and high efficiency.

FIG. 1 shows the construction method of plasmid pCRB200. FIG. 2 shows the construction method of plasmid pCRA820. FIG. 3 shows a method for constructing a plasmid pCRA703 for disrupting the PEPC gene. FIG. 4 shows a substance production metabolic pathway from glucose. FIG. 5 shows (A) glucose consumption rate (g / l / h) and (B) lactate production rate of Corynebacterium glutamicum R and Corynebacterium glutamicum R / R-nGAP strains after 24 and 48 hours. g / l / h). FIG. 6 shows Corynebacterium glutamicum R / ΔPEPC, Corynebacterium glutamicum R / ΔPEPC / R-nGAP, and Corynebacterium glutamicum R / ΔPEPC / Actino-nGAP, after 24 and 48 hours of the strain. (A) Glucose consumption rate (g / l / h) and (B) Lactic acid production rate (g / l / h) are shown.

Claims (6)

  A culture solution under anaerobic conditions using Corynebacterium glutamicum transformed with a DNA comprising the nucleotide sequence of SEQ ID NO: 7 or 16 encoding glyceraldehyde 3-phosphate dehydrogenase under an expressible control sequence It is characterized by accumulating lactic acid by increasing the metabolic rate of a saccharide selected from the group consisting of glucose, cellobiose, sucrose, lactose, maltose, dextrin, and soluble starch, and recovering lactic acid from the culture solution A method for producing lactic acid. Corynebacterium glutamicum, Corynebacterium glutamicum (Corynebacterium glutamicum) R FERM P- 18976, ATCC13032, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13655, ATCC13745, ATCC13746, ATCC13761, ATCC14020, and ATCC3183 1 or we selected The method for producing lactic acid according to claim 1, wherein the bacterium is any one of the fungi described above.   The transformed Corynebacterium glutamicum is Corynebacterium glutamicum R / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20875). Of producing lactic acid.   The transformed Corynebacterium glutamicum is Corynebacterium glutamicum R ΔPEPC / R-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20878) The method for producing lactic acid according to 1.   Claims wherein the transformed Corynebacterium glutamicum is Corynebacterium glutamicum R ΔPEPC / Actino-nGAP strain (National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM P-20877) The method for producing lactic acid according to 1.   The method for producing lactic acid according to claim 1, wherein the redox potential of the culture solution under anaerobic conditions is -200 millivolts to -500 millivolts.

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