BRPI0510919B1 - METHODS FOR THE PRODUCTION OF SUCCINITIC ACID AND POLYMER CONTAINING IT - Google Patents

Description

METHODS FOR. PROCEDURE OF SUCCYNIC ACID AND POLYMER CONTAINING IT Technical Field The present invention relates to the fermentation industry and a process for efficiently producing succinic acid by a fermentation method using a coryneform bacterium. Background Art For the production by fermentation of organic non-amino acids, including succinic acid, anaerobic bacteria such as those of the genera Anaerobiospirillum or Actinobâcusus (Patent Document 1 or 2 and Non-Patent Document 1) are generally used. The use of anaerobic bacteria increases the yield of the products, while these bacteria require many nutrients for their proliferation, so there is a need to add a large amount of organic nitrogen sources to the environment, such as corneal ICSL - Corn Steep Liquor). Adding abundant amounts of organic nitrogen sources not only leads to an increase in the cost of the medium, but also increases the cost of purification to isolate the product, which is not economical.

In addition, a method of cultivating aerobic bacteria such as corineform bacteria under aerobic condition is known for bacterial cells to proliferate, and then collecting and washing the cells to use them as resting bacteria to produce non-aerated succinic acid. Oxygen (Patent Documents 3 and 4}. This method is economical because bacterial cells can grow sufficiently in a simple medium to which a small amount of organic nitrogen * is added for bacterial cell proliferation; needs to be improved in terms of the amount of succinic acid that is generated, its concentration and its rate of production per bacterial cell, as well as in terms of simplifying the production process, and the like.

Furthermore, when aerobic bacteria such as coryneform bacteria are grown under conditions where oxygen is limited, organic acids in addition to the desired substance, such as lactic acid and acetic acid, accumulate in excess as byproducts, resulting in repression of the growth of the bacteria. bacterial cells and a significant decrease in fermentation productivity. In addition, excessive amounts of counter ions are required to neutralize organic acids generated as by-products, resulting in an uneconomical method. To address these problems, the reduction of lactate generated as a byproduct has been accomplished using a coryneform bacteria with reduced lactate dehydrogenase activity (Patent Document 5).

Even if the coryneform bacteria with reduced lactate dehydrogenase activity mentioned above are used, a large amount of acetic acid is generated as a byproduct. As a means of achieving acetic acid reduction in a culture medium, the following are known: a method for increasing the expression of an acetic acid-mimicking gene (aceP) in a bacterium belonging to the genus Escheríchia (Patent Document 6); a method for enhancing expression of a gene encoding ACE protein in a bacterium belonging to the genus Escherichia (Patent Document 7), and similar methods. These methods are intended to reduce the generation of acetic acid as a by-product by actively assimilating acetic acid released into a culture medium. At the same time, as methods for suppressing acetic acid generation as a by-product by suppressing acetic acid biosynthesis are known: a method for producing succinic acid using Escherichia coli, in which phosphoacetyltransferase (pta) and lactate dehydrogenase (ldh) are deficient (Patent Document 8); a method for producing an amino acid using an enterobacterium where pyruvate oxidase (poxB) is deficient, and a method for producing D-pantothenic acid using an enterobacterium where pyruvate oxidase (poxB) is deficient (Patent Document 9), How Enzymes responsible for the assimilation of acetic acid in coryneform bacteria have been reported to acetate kinase, (ack) and phosphotransacetylase (pta) {Non-Patent Document 2}. On the other hand, it is assumed that not only the enzymes mentioned above, but also a plurality of enzymes such as pyruvate oxidase (poxB) (Patent Document 10), acylphosphatase (acp), aldehyde dehydrogenase and acetyl CoA hydrolase are responsible for the generation. of acetic acid, but a specific enzyme contributing to the synthesis of acetic acid has not been clarified. Therefore, a method for producing succinic acid using a strain of a coryneform bacterium that has a reduced acetic acid biosynthetic enzyme is not known.

Acetyl-CoA hydrolase is an enzyme for generating acetyl-CoA acetic acid and water (3.1.2.1), and the gene sequence in Corynebacterium glutamicum has been predicted (Patent Document 11). However, there are no reports of gene cloning and gene expression analysis, and their actual function is unclear.

Patent Document 1: US Patent No. 5,143,833 Patent Document 2: US Patent No. 5,504,004 Patent Document 3: JP11-113588A Patent Document 4: JP11-196888A Patent Document 5: JP11-206385A Patent 6: JP06-14781A

Patent Document 7: JP07-67683A Patent Document 8: WO 95/06532 Patent Document 9: WO-02/36797 Patent Document 10: EP1096013A

Patent Document 11: EP1108790A

Non-Patent Document 1: International Journal of Systematic Bacteriology, vol.49, p 207-216, 1999 Non-Patent Document 2: Microbiology. 1999, Feb; 145 (Pt2): 503-13 Description of the Invention An object of the present invention is to provide a coryneform bacterium that is better able to produce succinic acid.

The inventors of the present invention have worked hard to solve the problems mentioned above and, as a result, have found that the ability to produce succinic acid improves with decreased acetyl CoA hydrolase activity in a coryneform bacterium. In addition, they have found that the generation of acetic acid as a by-product is reduced by decreasing phosphotransacetylase and acetate kinase activities in addition to acetyl CoA hydrolase activity and thus embodied the present invention.

That is, the present invention is as follows: (1) A coryneform bacterium which is capable of producing succinic acid, said bacterium being modified such that the activity of acetyl CoA hydrolase is decreased. (2) there are coryneform bacteria according to (1), wherein acetyl-CoA hydrolase is a protein as described in (A) or (B) below: (A) a protein having an amino acid sequence of SEQ ID NO: 45; OR (B) a protein having an amino acid sequence of SEQ 1D NO; 45, including substitution, suppression, insertion or addition of one or more amino acids, and having acetyl CoA hydrolase activity. (3) The coryneform bacterium according to (1) or (2), wherein acetyl-CoA hydrolase activity has been decreased by disruption of an acetyl-CoA gene on a chromosome. (4) The coryneform bacterium according to (3), wherein the acetyl CoA hydrolase gene is a DMA as described in (a) or (b) below; (a) a DNA consisting of a nucleotide sequence of nucleotides number 103? at 2542 in SEQ ID NO: 44; or (b) a DNA that hybridizes to a nucleotide nucleotide sequence number 1037 to 2542 in SEQ ID NO; 44 or a probe that can be prepared from the nucleotide sequence under stringent conditions, and which encodes a protein that has acetyl CoA hydrolase activity. (5) The coryneform bacterium according to either of (1) to (4), which has been further modified so that the activities of one or both phosphotransacetylase and acetate kinase have been decreased. (6) The coryneform bacterium according to any one of (1) to (5), said bacterium being further modified such that lactate dehydrogenase activity has been decreased. (7) The coryneform bacterium according to any one of (1) to (61), wherein said bacterium has been further modified so that pyruvate carboxylase activity has been increased. {81 One method for producing succinic acid consisting of allowing that the coryneform bacteria according to any one of (1) to (7), or a treated product thereof, act on an organic raw material in a reaction liquid containing ion carbonate, lon bicarbonate, or carbon dioxide to generate and accumulating succinic acid in the reaction liquid, and in collecting succinic acid from the reaction liquid, (9) The production method according to [8), wherein the coryneform bacterium or its treated product is allowed to act on the organic raw material under anaerobic conditions. (10) A method for producing a succinic acid-containing polymer, consisting of the stages of producing succinic acid by the method according to (8) or (9) and polymerizing the succinic acid obtained.

Brief Description of the Drawings Figure 1 shows the procedures for construction of plasmid pBS3. Figure 2 shows the procedures for construction of plasmid pBS45. Figure 3 shows the procedures for constructing plasmid pBSST. Figure 4 shows the procedures for constructing plasmid pldh56-1. Figure 5 shows the procedures for constructing plasmid pBSST :: Aack. Figure 6 shows the procedures for constructing plasmid pBSST :: Apta-ack. Figure 7 shows the procedures for constructing plasmid pBSST :: ΔροχΒ. Figure 8 shows the procedures for constructing plasmid pBS4S :: ãach.

Description of Preferred Embodiments Hereinafter, embodiments of the present invention will be described in detail herein. <1> Corineform bacteria to be used in the present invention In the present invention, the term "corineform bacteria" includes a bacterium that had been classified as genus Brevibacterium, but is now classified as genus Corynebacterium {Xnt. J. Syst. Bacteriol., 41, 255 (1981)), and also includes a bacterium belonging to the genus Brevibacterium, which is closely related to Corynebacterium. Examples of said coryneform bacteria include the following.

Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium tbermoaminogenes Corynebacterium herculis Brevibacterium divaricatum Brevibacterium spun Brevibacterium irmariophilim Brevibacterium lactofermentum Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes Brevibacterium album Brevibacterium selinum Microbacterium ammoniapbilum In the present invention, the term "ability to produce Succinic acid "means an ability to accumulate succinic acid in a medium when culturing the coryneform bacteria of the present invention. The ability to produce succinic acid may be an inherent feature of a wild type coryneform bacterium, or a feature conferred by cultivation.

To confer the ability to produce succinic acid by cultivation, methods that have been employed in the cultivation of corynefome bacteria may be applied, which include the acquisition of mutant strains for metabolic regulation, the creation of a recombinant strain having a biosynthetic enzyme for a substance. desired ■ increased, & similar [Amino Acid Fermentation, Japan Scientific Societies Press, first edition published May 30, 1986, p77-100). In such methods one or two or three or more characteristics such as mutations in metabolic regulation and augmentation of biosynthetic enzymes for a desired substance may be conferred. Providing properties such as mutations in metabolic regulation and increased biosynthetic enzymes can be combined.

Particularly preferred specific examples of a coryneform bacterium that is capable of producing succinic acid include Brevi ba ct and ri a flavum strain with decreased lactate dehydrogenase activity {JP11-206305A}; Brevibacterium flavum strain MJ233 / pPCPYC with increased pyruvate carboxylase or phosphoenol pyruvate carboxylase activity [WO 01/27258 and JP11-136887A); Brevibacterium flavum MJ-233 (FERM BP-1497); Brevibacterium flavum MJ-233 (FERMPB-1498); Brevibacterium ammoniagenes ATCC6872; Corynebacterium glutamicum ATCC31831, and Brevibacterium lactofermentum ATCC13869. As currently Brevibacterium flavum can be classified as Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., I.udwig, W. and Schleifer, KH, International Journal of Systematic Bacteriology, 1991, vol. 41, p255-260), said Brevibacterium flavum strain MJ-233 and its mutant strain MJ-233 AB-41 are defined as the same lines as Corynebacterium glutamicum strain MJ-233 and Corynebacterium glutemlcum strain MJ233 AB-41, respectively. <2> Construction of the coryneform bacterium of the present invention The coryneform bacterium of the present invention has the above mentioned ability to produce succinic acid and has been modified such that the activity of acetyl CoA hydrolase has been decreased.

In the cultivation of bacteria. According to the present invention, provision of the ability to produce succinic acid and modification to decrease acetyl CoA hydrolase (EC 3.1.2.1) activity can be performed in any order. The term "acetyl CoA hydrolase (ACH) activity" means an activity to catalyze the reaction to generate acetic acid from acetyl CoA and water. The term "modified such that acetyl-CoA hydrolase activity is decreased" means that acetyl-CoA hydrolase activity is decreased compared to specific activity of an unmodified lineage, such as wild-type corynema bacteria. ACH activity is preferably reduced to 50% or less per bacterial cell, more preferably 30% or less, even more preferably 10% or less per bacterial cell, as compared to an unmodified strain. In this case, examples of a wild-type coryneform bacterium to be used as a control include Brevibacterium lactofermentam ATCC13869 (wild-type strain) and Brevibacterium lactofermentam Aldh strain (unmodified strain). Acetyl CoA hydrolase activity can be determined according to the method of Gergely, J. et al. (Gergely, J., Hele, P. & Ramkrishnan, CV (1952) J. Biol. Chem. 198 p323-334) . The term "decreased" includes the total loss of activity. The coryneform bacterium of the present invention preferably has a lower acetyl CoA hydrolase activity than that of a wild type or unmodified strain and more preferably has better accumulation of succinic acid compared to said strains.

Examples of acetyl CoA hydrolase having the activity mentioned above include a protein having an amino acid sequence of SEQ ID NO: 45. In addition, it may be a protein having an amino acid sequence of SEQ ID NO: 45 including a substitution, deletion, insertion or addition of one or more amino acids as long as it has an acetyl CoA hydrolase activity. In this case, for example, the term "various" means from 2 to 20, preferably from 2 to 10, more preferably from 2 to 5. The term "modified such that acetyl CoA hydrolase activity is decreased" includes decrease number of acetyl-CoA hydrolase molecules per cell, decreased acetyl-CoA hydrolase activity per molecule, and the like. Specifically, this is accomplished by making a gene encoding acetyl-CoA hydrolase in a chromosome deficient by modifying an expression regulatory sequence, such as a promoter or Shine-Dalgarno (SD) sequence, or the like. Examples of the acetyl-CoA hydrolase gene on a chromosome include a DNA having a nucleotide nucleotide sequence number 1037 to 2542 of SEQ ID NO: 44. It may be a DNA that hybridizes to nucleotide nucleotide sequence number 1037 to 2542 of SEQ ID NO: 44 or a probe which may be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a protein that has acetyl CoA hydrolase activity. the term "stringent conditions" means conditions under which the so-called specific hybrid is formed and the non-specific hybrid is not formed. Conditions are difficult to define clearly by numerical values, but examples thereof include conditions which consist of washing once, preferably - twice or three times at an exit concentration corresponding to 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS at 60 ° C. The acetyl CoA hydrolase gene (hereinafter referred to as the ach gene) can be cloned by synthesizing synthetic oligonucleotides based on a GenBank-recorded CoryneJhacterium glutamlcum sequence (NCB22480 GenBank Accession No. NC_003450, 2730884 of NC_003450)), and performing a PCR using a Corynebacteri chromosome glutamicum as a templafce. In addition, a sequence of a coryneform bacterium such as Brevibacterium lactofermentum with a nucleotide sequence determined by recent genome design can also be used. Chromosomal DNA can be prepared from a bacterium as a DNA donor, for example by the Saito and Miura method (H. Saito and K. Miura, Brochem. Biophys. Acta, 72, 619 (1963), Experimental Manual for Biotechnology, published by the Biotechnology Society, Japan, p97-98, Baifukan Co., Ltd., 1992) or the like. The ach gene prepared in this manner, or a portion thereof, may be used for gene disruption. A gene to be used for gene disruption only needs to have sufficient homology to cause a homologous recombination with an ach gene to be disrupted in the chromosomal DNA of a coryneform bacterium (for example, a gene having the nucleotide sequence number 1037 to 2542 in SEQ ID NO: 44), and thus said homologous gene may also be used. In this case, the homology which is sufficient to cause homologous recombination is preferably not less than 70%, more preferably not less than 80% and even more preferably not less than 90%. In addition, a DNA capable of hybridizing to the above mentioned gene under stringent conditions may cause homologous recombination. The term "stringent conditions" refers to conditions in which a so-called specific hybrid is formed and a non-specific hybrid is formed. Conditions are difficult to define clearly by numerical values, but conditions include, for example, conditions consisting of washing once, preferably twice or three times at salt concentrations corresponding to 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS at 6 ° C.

For example, using the gene mentioned above, a suppressed form of the ach gene is prepared, which is modified so as not to produce acetyl-CoA hydrolase that normally functions by suppressing a partial sequence of the ach gene, and transforms it. a coryneform bacterium with a gene-containing DNA to cause recombination between the suppressed form of the gene and the gene on a chromosome, thereby breaking the ach gene on a chromosome. Said gene disruption by gene replacement using homologous recombination has already been established, and examples thereof include a method using a linear DNA and a method using a plasmid containing a temperature sensitive origin of replication (US Patent No. 6,303. 383, or JP05-0Ü7491A). In addition, the aforementioned gene disruption based on gene replacement using homologous recombination can also be performed using a plasmid that has no replication capability in a host.

For example, an ach gene on a host chromosome may be replaced by a suppressed form of ach gene according to the following procedures. First, a plasmid is prepared for recombination by inserting a temperature-sensitive origin of replication, the suppressed form of the ach gene, the 3acB gene that encodes levansucrase, and a marker gene that demonstrates resistance to a drug such as chloramphenicol, In this case, the sacB gene encoding ievansucrase is a gene that is used to efficiently select a lineage in which a portion of the vector has been excised from a chromosome (Schafer, A. et al., Gene 145 (1994) 69-73 ), That is, when Ievansucrase is expressed in a coryneform bacterium, the levan generated by sucrose assimilation acts lethally on the bacterium and thus the bacterium cannot grow. Therefore, if a bacterial strain in which a vector carrying Ievansucrase remains on the chromosome is grown on a sucrose-containing plate, it cannot grow. The result is that only a bacterial strain from which the vector has been extirpated can be selected on the plaque containing sucrose.

Genes having the following sequences can be used as a sacB gene or homologous gene thereof, Bacillus subtilisx sacB GenBank Accession Number XO2730 (SEQ ID NO: 35) Bacillus amyloliquefaciensx sacB GenBank Accession Number X52988 Zymomonas mobilist sacB Accession Number GenBank L33402 Bacillus stearothermophilus: surB GenBank Accession Number U34874 Lactojbacillus sanfrancíscensis: frfA Genbank Accession Number AJ508391 Acetobacter xylins: IsxA GenBank Accession Number AB034152 Giuconacetobacter diazotropied4 Bacterium oData Bacterial cDNA istransformed GenBank The transformation may be performed according to a transformation method that has been previously reported. Examples of methods include a method for increasing the permeability of a DMA by treating the cells of a recipient bacteria with calcium chloride, as reported with reference to Escherichia coli K-12 (Mandei, M. and Higa, A. J, J. Mol. Biol., 53,159 (1970)) and a method for preparing competent cells using proliferative cells for DNA introduction, as reported with reference to Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 ( 1977)). Alternatively, as reported with reference to Bacillus subtilis, actinomycetes and yeast, a method for introducing recombinant DNA into cells of a DNA recipient bacterium (Chang, S. and Choen, SN, Molec. Gen. Genet. , 168, 111 (1979)); Bibb, M.J., Ward, J.M. and Hopwood, O.A., Wature, 274, 398 (1978); Hinnen, A., Hicks, J.B., and Fink, G. R., Proc. Natl. Acad. Know. USA, 75, 1929 (1978)). In addition, a coryneform bacteria can be transformed by the electric pulse method (Sugimoto et al., JP02-207791A).

Examples of a temperature sensitive plasmid for a coryneform bacterium include p48K and pSFKT2 (JP2000-262288A) and pHSC4 (French Patent No. 2667875 (1992) and JP05-7491A). These plasmids may be autonomously replicated in a coryneform bacterium at least 25 ° C, but are not autonomously replicable at 37 ° C. Escherichia coli AJ12571 with pHSC4 was deposited with FERH Accession Number P-11763 at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, (now International Patent Depositary), National Institute of Advanced Industrial Science and Technology (Centrai 6, 1 -1-1, Kigashi, Tsukuba, 1 bafei, 305-5466, Japan), on October 11, 1990, and was then transferred to an international deposit in accordance with the provisions of the Budapest Treaty on 26 October 1990. August 1991, under FERM Access Number 3P-3524.

A transformant obtained as described above is cultured at a temperature at which the temperature sensitive origin of replication does not function (25 ° C) to thereby obtain a strain into which the plasmid was introduced. The strain into which the plasmid was introduced is grown at an elevated temperature to excise the temperature sensitive plasmid, and the bacterial strain is applied to a plate containing an antibiotic. The temperature sensitive plasmid cannot replicate at an elevated temperature. Therefore, a bacterial lineage from which the plasmid has been deleted cannot grow on a plate containing an antibiotic, but a bacterial lineage in which recombination has occurred between the plasmid de ach gene and the ach gene on a chromosome appears with a very low frequency.

In the lineage obtained by introducing recombinant DNA into a chromosome as described above, recombination occurs with a sequence of the ach gene that was originally present on one chromosome, and two fc genes of the ach chromosomal gene and the deleted form of gene. ach are inserted into a chromosome so that other portions of the recombinant DNA (vector part, temperature-sensitive origin of replication, and drug-resistant marker) are present between the fusion genes.

So, in order to leave only the suppressed form of the ach gene in a chromosomal DNA, the gene is deleted along with the vector portion (the origin of temperature sensitive replication and drug resistant marker) of the chromosome. This procedure causes a situation where the normal ach gene remains in the chromosomal DNA and the suppressed form of the ach gene is extirpated, or instead a situation where the normal ach gene is deleted and the suppressed ach gene form remains in the DNA chromosomal. In either case, when culturing is performed at a temperature that allows a temperature-sensitive origin of replication to function, the cleaved DNA is kept in a cell as a plasmid. Then, when culturing at a temperature that does not allow a temperature sensitive origin of replication to function, the ach gene in the plasmid is deleted from the cell along with the plasmid. Thus, a strain in which the suppressed form of ach gene remains on the chromosome is selected by PCR, Southern hybridization or the like to thereby produce a strain in which the ach gene is disrupted.

If a plasmid that is used is used. Having no replicability in a coryneform bacterium instead of the temperature-sensitive plasmid mentioned above, gene disruption can also be performed in a similar manner. The plasmid that has no replicability in a coryneform bacterium is preferably a plasmid that has replicability in Escherichia coli, and examples thereof include pHSG299 (Takara Bio Inc.) and pHSG399 (Takara Bio Inc.). One method of decreasing acetyl-COa hydrolase activity includes not only the genetic engineering method cited above, but also a method of treating a coryneform bacterium with ultraviolet irradiation or. a commonly used mutagen for mutation, such as N-methyl-N'-nitro-N-nitrous guanidine (NTG) and nitrous acid, and select a bacterial strain that has reduced acetyl CoA hydrolase activity. In the present invention, It is more effective to use a modified bacterial strain such that one or both of the phosphotransacetylase (hereinafter called PTA) and acetate kinase (hereinafter called ACK) activities are decreased, in addition to decreased ACH activity.

In the present invention, phosphotransacetylase (PTA) activity means an activity to catalyze a reaction to generate acetyl phosphate by transferring phosphate to acetyl CoA (EC: 2,3,1,8), and acetate kinase (ACK) means an activity to catalyze a reaction. reaction to generate acetic acid from acetyl phosphate and ADP (EC: 2,7.2.1) - Reduction of these activities may be achieved by disruption of the genes coding for the enzymes mentioned above or by modification of a regulatory sequence expression, such as a promoter and Shine-Dalgarno (SP) sequence of the genes encoding the enzymes. Gene disruption can be performed in the same manner as the above method for ach gene disruption.

As genes encoding the enzymes, for example, the following Corynebacterivm glütamicum genes deposited in GenBank can be used: pta (phosphoacetyltransferase) gene: GenBank Accession No. NCgl2657 (nucleotide complementary line numbers 2936506-2937495 in NC 003450) ( nucleotide numbers 956-1942 in SEQ ID NO: 39).

Ack (acetate kinase) gene: GenBank Accession No. NCgll2656 (complementary nucleotide line numbers 2935323-2936506 in NC 003450) (nucleotide numbers 1945-3135 in SEQ ID NO: 39). The expression "phosphotransacetylase activity (hereinafter hereinafter referred to as PTA) is decreased" means that the die PTA activity is decreased compared to an unmodified PTA strain. PTA activity is lower than that of a non-PTA strain. It is modified with respect to PTA or a wild-type strain, and is preferably reduced to 501 or less per bacterial cell, more preferably 10% or less per bacterial cell. PTA activity can be completely eliminated. Decreased PTA activity can be confirmed by determining PTA activity by a method of Klotzsch et al. (Klotzsch H.R., Meth Enzymol. 12, 381-386 (1969)). A coryneform bacterium in which both ACH and PTA activities are decreased can be obtained by constructing a corineform bacterium having decreased ACH activity and modifying it to decrease PTA activity. However, there is no preference as to the order for modification to decrease PTA activity and modification to decrease ACH activity. The expression "acetate kinase activity (hereinafter referred to as ACK) is decreased" means that activity ACK activity is decreased compared to a wild type or unmodified lineage with reference to ACK. ACK activity is lower than that of an unmodified lineage with respect to ACK or a wild type lineage and preferably decreases to 50% or less per bacterial cell, more preferably 10% or less per bacterial cell compared to an unmodified ACK strain. ACK activity can be completely eliminated. Decreased ACK activity can be confirmed by determining ACK activity by a method of Ramponi et al. (Ramponi <3., Meth. Enzymol. 42, 409-426 (1975)). As a coryneform bacterium in which both ACH and ACK activity are decreased can be obtained by constructing a corineform bacterium with decreased ACH activity and modifying it to decrease ACK activity. However, there is no preference for the order of performing modification to decrease ACK activity and modification to decrease ACH activity.

In the present invention, it is more effective to use a modified bacterial strain so that lactate dehydrogenase activity (hereinafter referred to as LDH) is decreased, in addition to decreasing said ACH activity. Lactate dehydrogenase activity means an activity to catalyze a reaction to generate lactic acid by reducing pyruvic acid using NADH as a coenzyme. The expression "lactate dehydrogenase activity is decreased" means that LDH activity is decreased compared to that of an unmodified LDH strain. LDH activity is lower than that of an unmodified lineage with respect to LDH, or a wild type lineage, and is preferably reduced to 50% or less per bacterial cell, preferably 10% or less per bacterial cell. LDH activity can be completely eliminated. The decrease in LDH activity can be confirmed by determining LDH activity by a method of L. Kanarek et al. (L. Kanarek and R.L. Hill, J. Biol. Chem. 239, 4202 (1964)). A coryneform bacterium of the present invention may be obtained by preparing a coryneform bacterium with decreased LDH activity and modifying it to decrease ACH activity. However, there is no preference as to the order of modification to decrease LDH activity and modification to decrease ACH activity.

As an Idh gene, for example, a gene having a sequence represented by SEQ 1D NO: 37 can be used, and the gene disruption can be performed similarly to that of the above ach gene.

Moreover, in the present invention, a modified bacterium can be used such that pyruvate carboxylase activity (hereinafter referred to as PC) is increased, in addition to decreased ACH activity. The term "pyruvate carboxylase activity is increased" means that PC activity is increased compared to that of a wild type strain or an unmodified strain such as a parent strain. PC activity can be determined by a method of Peters-Wendisch, p.G and others. (Peters-Wendisch, P.G- et al.

Microbiology 143, 1095-1103 (1997)).

As a PC gene which encodes a PC protein to be used in the method of the present invention, a gene whose nucleotide sequence has been determined may be employed, or a gene obtained by isolating a DMA fragment encoding a protein with activity. PC from a chromosome of microorganisms, animals, plants or the like, according to the method described below and determining its nucleotide sequence. In addition, after nucleotide sequence determination, a synthesized gene based on the sequence can be used. For example, a Corynebacterium glutamicum pyruvate carboxylase gene ATCC13032 (GenBank Accession No. NCgl0659: SEQ ID NO: 46) may be used. In addition, PC genes derived from the following Human organisms [Biochem. Biophys, Res. Comm., 202, 1009-1014, (1994)] Mouse [Proc, Natl. Acad, I know. USA, 90, 1766-1779, (1993)) Rat [GENE, 165, 331-332, (1995) 1 Yeast; Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315, (1991)] Scbizosaccharomyces pombe [No. Access DDBJ; D78170] Bacillus stearothermophilus [GENE, 191, 47-50, (199 ')] Ehizobium etli [J, Bacteriol., 178, 5960-5970, (1996)) A DNA fragment containing a PC gene can be expressed by inserting the DNA fragment into a suitable expression plasmid such as pUC118 (Takara Bio Inc.), and introducing it into a suitable host microorganism such as Escherichia coli JM109 (Takara Bio Inc.) * The product of gene expression of PC, which is pyruvate carboxylase, can be confirmed by determining PC activity by the known method described above in the transformant and then comparing the PC activity determined with the PC activity of a extracted crude enzyme solution. of a non-transforming lineage. The DNA fragment containing the PC gene is inserted into a suitable plasmid, such as a plasmid vector containing at least one gene responsible for the plasmid replication function in coryneform bacteria; In this way, a recombinant plasmid capable of high PC expression in coryneform bacteria can be obtained. Here, in the recombinant plasmid, a promoter for PC gene expression may be a promoter of coryneform bacteria. However, it is not limited to said promoter and any promoter may be used provided it has a nucleotide sequence capable of initiating transcription of the PC gene.

Plasmid vectors into which the PC gene may be introduced are not limited as long as they contain at least one gene responsible for replication function in coryneform bacteria. Specific examples thereof include: plasmid pCRY30 described in JP03-210184A; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX, each being described in JP02-72876A and US Patent No. 5,185,262; plasmids pCRY2 and PCRY3, each described in JP01-191686A; pAM330 described in JP58-67679A; pHM1519 described in JP58-77895A; pAJ655, pAJ611, and pAJ188, each described in JP58-192900A; pCG1 described in JP57-1345QOA; pCG2 described in JP58-3S197A, and pCGG4 and pCG 11, each described in JP57-133799A.

Among these, the plasmid vectors used in the host-vector system for coryneform bacteria are preferably those having a gene responsible for the plasmid replication function in corineform bacteria and a gene responsible for the plasmid stabilization function in corineform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX may preferably be used.

Corineform bacteria having enhanced expression of the PC gene can be obtained by transforming a corineform bacteria, for example Brevibacterium lactofermentum, lineage 2256 (ATCC138 69) with a recombinant vector prepared by inserting the PC gene into an appropriate site of a plasmid vector. that is replicable in aerobic coryneform bacteria as described above. Transformation may be conducted, for example, by the electric pulse method (Irs, Microbiol., Vol. 144, p. 181-185, 1993). PC activity can also be increased by enhancing gene expression by introducing, substituting, amplifying or the like the PC gene on a chromosome by a known method of homologous recombination. By breaking the ach gene into a strain with high expression of the PC gene, a bacterial strain with increased PC activity and decreased ACH activity can be obtained. There is no preference in the order in which modifications are made to decrease ACH activity and increase PC activity.

Furthermore, in the present invention, a bacterium that is modified such that ACH activity, or ACH, PTA and ACK activities is / is decreased and further modified so that LDH activity is decreased and PC is increased will be used especially effectively for the production of succinic acid. Succinic acid production using the bacterium of the present invention Succinic acid can be efficiently produced by cultivating the coryne bacteria thus obtained in a medium to produce and accumulate succinic acid in the medium and collect succinic acid from the medium.

With reference to the use of the bacteria mentioned above in a reaction to produce succinic acid, the bacteria subjected to culture on slanting solid medium such as an agar medium may be used directly for the reaction, but one may preferably use a bacterium obtained by cultivating it. previously the bacteria mentioned above in a liquid medium (seeded culture). Succinic acid can be produced by allowing the bacteria obtained in the seeded culture to react with an organic material while the bacteria reproduce in a medium containing the organic raw material. In addition, succinic acid can also be produced by harvesting proliferating bacterial cells and then reacting the bacterial cells with an organic raw material in a reaction liquid containing the organic raw material. Furthermore, for the purpose of using an aerobic coryneform bacterium in the method of the present invention, it is preferable to use the corineform bacterium for reaction after cultivating the bacterium under normal aerobic condition. The medium to be used for culture may be any medium normally used for culturing microorganisms. For example, conventional media which can be prepared by adding sources of natural nutrients such as meat extract, yeast extract and peptone to a composition made of inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used. In case of collection and use of the bacterial cells after culture, the bacterial cells are harvested by centrifugation, membrane separation or the like and then used for the reaction.

In the present invention, a treated bacterial cell product may also be used. For example, treated bacterial cell products include immobilized bacterial cells that are immobilized to acrylamide, carrageenan or the like, disrupted bacterial cells, centrifugation supernatant thereof, or fractions obtained by partial purification of the supernatant with ammonium sulfate treatment or similar. The organic raw material to be used in the production method of the present invention is not particularly limited as long as it is a carbon source that can be assimilated by the microorganism described herein to produce succinic acid. In general, a fermentable carbohydrate, including a carbohydrate such as galactose, lactose, glucose, fructose, glycerol, saccharin, sucrose, starch and cellulose, is used; a polyalcohol such as glycerin, mannitol, xylitol and ribitol. Of all these, glucose, fructose and glycerol are preferable and glucose is especially preferable.

In addition, a saccharified, molten starch liquid or the like which may contain any of the fermentable carbohydrates mentioned above may also be used. Any of those fermentable carbohydrates may be used alone or in combination. The concentration at which the above-mentioned organic raw material is used is not particularly limited, but it is advantageous to increase the concentration as much as possible within the range that does not inhibit succinic acid production. The reaction is usually carried out in the presence of organic raw material in the range of 5 to 301 (weight / volume), preferably 10 to 20% (w / v). * Organic raw material may be additionally added as the raw material decreases. As mentioned above, as the reaction progresses, the reaction liquid containing the organic raw material is not particularly limited. The reaction liquid to be used may be water, buffer, medium or the like, but the medium is most preferable. The reaction liquid is preferably a liquid containing a nitrogen source, inorganic salts and the like. Here, the nitrogen source is not particularly limited as long as it can be assimilated by the microorganism described herein to produce succinic acid. Specific examples of the nitrogen source include various organic and inorganic nitrogen compounds, such as ammonium salts, nitrate, urea, soy hydrolyzate, casein hydrolyzate, peptone, yeast extract, meat extract and myococcus. Examples of inorganic salts include various types of phosphoric acid salts, sulfuric acid salts, and metal salts of magnesium, potassium, manganese, iron, zinc and the like. In addition, components that promote the growth of bacterial cells may be added as necessary, including: vitamins such as biotin, pantothenic acid, inositol and nicotinic acid; nucleotides, and amino acids. Further, it is preferable that an optimal amount of a commercially available antifoam agent be added to the reaction liquid to prevent foaming at the time of reaction. The pH of the reaction liquid may be adjusted by the addition of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, or the like. The pH for the reaction is generally from 5 to 10, preferably from 6 to 9.5, so the pH of the reaction solution may be adjusted within the above range with alkaline, carbonate, urea or the like during the reaction, if this is the case. is necessary.

Preferably, the medium contains ion carbonate, ion bicarbonate or carbonic acid (carbon dioxide) gas. Ion carbonate or ion bicarbonate is provided by magnesium carbonate, sodium carbonate, potassium carbonate or potassium bicarbonate, which may also be used as a neutralizing agent. However, if necessary, ion carbonate or ion bicarbonate may also be provided by carbonic acid or bicarbonic acid, or salts thereof, or carbonic acid gas. Specific examples of the salts of carbonic acid or bicarbonic acid include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate and potassium bicarbonate. In addition, the ion carbonate or ion bicarbonate is added at a concentration of 0.001 to 5 M, preferably 0.1 to 3 M and more preferably 1 to 2 M. When the medium contains carbonic acid gas, the amount of carbonic acid gas to be contained is from 50 mg to 25 g, preferably from 100 mg to 15 g, more preferably from 150 mg to 10 g per liter of liquid. The optimum temperature at which the bacteria to be used in the reaction grows is usually in the range of 25 ° C to 35 ° C.

On the other hand, the temperature at the time of the reaction is generally in the range 25 ° C to 40 ° C, preferably in the range 30 ° C to 37 ° C. The amount of bacterial cells to be used in the reaction is not particularly limited, but the amount is adjusted within the range from 1 to 700 g / l, preferably from 10 to 500 g / l, and more preferably from 20 to 400 g / l. 1. The reaction time is preferably from 1 to 168 hours, more preferably from 3 to 72 hours.

For bacterial culture, it is necessary to provide ".......... oxygen by aeration and agitation. On the other hand, the reaction to produce succinic acid may be carried out with aeration and agitation, or may be performed under normal conditions. here, the term "anaerobic condition" means that the reaction is conducted while keeping the dissolved oxygen in the liquid low, in which case it is preferable to conduct the reaction with a dissolved oxygen level of 0 at 2 ppm, preferably from 0 to 1 ppm, and more preferably from 0 to 0.5 ppm. For this purpose, a method may be used wherein a container is hermetically sealed so that the reaction is carried out without aeration; a method in which an inert gas such as nitrogen gas is provided for carrying out the reaction, a method in which the reaction is performed by aeration with an inert gas containing carbonic acid gas, and similar methods. Acetic acid accumulated in the reaction liquid (culture solution) may be isolated from the reaction liquid and purified according to a conventional procedure. Specifically, succinic acid may be isolated and purified by removing solid materials, including bacterial cells, by centrifugation, filtration or the like, and desalting the solution with an ion exchange resin or the like, followed by crystallization or column chromatography. of the solution.

In the present invention, following the production of succinic acid by the method of the present invention as described above, a polymerization reaction is carried out using succinic acid obtained as the starting material to produce a succinic acid-containing polymer. The succinic acid-containing polymer may be a homopolymer or a copolymer with other polymeric raw materials. In recent years, environmentally friendly industrial products have been on the rise and polymers made from plant-based raw materials have attracted attention. The succinic acid to be produced in the present invention may be processed into polymers such as polyester and polyamide and then used. Specific examples of succinic acid containing polymers include a succinic acid containing polyester obtained by polymerization between a diol such as butanediol or ethylene glycol and succinic acid and a succinic acid containing polyamide obtained by polymerization between a diamine such as hexamethylenediamine and succinic acid. .

In addition, succinic acid or a succinic acid-containing composition obtainable by the production method of the present invention may be used as food additives, pharmaceuticals, cosmetics and the like.

Examples Hereinafter the present invention will be described in more detail by way of examples.

Example 1 <1> Construction of a disruption vector carrying a sacB gene (A) Construction of pBS3 The sacB gene (SEQ ID HO: 35) was obtained by PCR using a Sacillus subtilis chromosomal DNA as template and SEQ. ID NOS, * 1 and 2 as primers. PCR was conducted using LA Taq (Takara Bio Inc.), so that a thermal retention cycle was performed at 94 ° C for 5 minutes and then a denaturation cycle at 94 ° C for 30 seconds, annealing at 100 ° C. 49 ° C for 30 seconds and stretching at 72 ° C for 2 minutes was repeated 25 times. The PCR product thus obtained was purified by a standard procedure and then digested with Bglix and BamHI, and then the ends were trimmed. The fragment was inserted into a pHSG299 site that had been digested with "Avall and whose ends had been trimmed. Escherichia coli JM109 competent cells (Takara Bio Inc.) were used for transformation with this DNA and the transformed cells were applied to a LB medium containing 25 μ9 / ιη1 of kanamycin (hereinafter abbreviated as Km), followed by overnight culture Subsequently, the colonies that appeared were harvested and single colonies were isolated and thus transformants were obtained. of the transformants and a plasmid into which a PCR product of interest was inserted was called pBS3 Figure 1 shows the procedures for the construction of pBS3.

(B) Construction of pBS4S The Smal site in the sequence of the kanamycin resistance gene present in pBS3 was disrupted by cross-PCR mediated nucleotide substitution without causing any amino acid substitution to obtain a plasmid. First, PCR was performed using pBS3 as a therapist and synthetic DNfts of SEQ 1D Nos: 3 and 4 as primes, and thereby the extended product of the N-terminal region of the kanamycin resistance gene was obtained. On the other hand, to obtain the extended C-terminal region product of the Km resistance gene, PCR was performed using pBS3 as a therapist and synthetic DNAs of SEQ ID Nos: 5 and 6 as primers. PCR was conducted using Pyrobest DNA Polymerase (Takara Bio Inc.), so that a thermal retention cycle was performed at 98 ° C for 5 minutes and then a denaturation cycle at 98 ° C for 10 seconds. , annealing at 57 ° C for 30 seconds and stretching at 72 ° C for 1 minute was repeated 25 times, thereby yielding a PCR product of interest. SEQ ID Nos: 4 and 5 are partially complementary to each other and the Smal site in the sequence is disrupted by nucleotide substitution without causing any amino acid substitution.

Then, to obtain a fragment of a mutant kanamycin resistant gene in which the Smal is disrupted, the N-terminal and C-terminal gene products of the above-mentioned kanamycin resistance gene were mixed at approximately equimolar concentration. , and PÇR was performed using the mixture of the genetic products as therapists and synthetic DNAs of SEQ ID NOS: 3 and 6 as primers, thereby producing the extended product of a mutation-introduced Km resistance gene. PCR was conducted using Pyrobest DNA Polymerase (Takara Bio Inc *} such that a thermal retention cycle was performed at 98Â ° C for 5 minutes and then a denaturation cycle at 98Â ° C for 10 seconds, annealing. at 57 ° C for 30 seconds and elongation at 72 * 0 for 1.5 minutes was repeated 25 times, thus yielding a PCR product of interest.The PCR product was purified by a standard procedure and then digested with BanII followed by insertion into the BanII site of pBS3 mentioned above Competent Eschericltiâ coli JM109 cells (Takara Bio Inc.) were used for transformation with this DNA and the transformed cells were applied to an LB medium containing 25 µg / ml kanamycin, followed by culture of the Subsequently the colonies that appeared were harvested, and single colonies were isolated, thereby obtaining transformants, plasmids were extracted from the transformants and a plasmid in which a PCR of interest was inserted was called pRS4S. Figure 2 shows the procedures for building pBS4S.

(C) Construction of pBS5T

A plasmid was constructed by inserting a temperature-sensitive origin of replication for a coryneform bacterium into pBS4S constructed as described in (B) above. That is, a temperature sensitive origin of replication .............. "temperature for a coryneform bacterium was obtained by digesting pHSC4 (JP05-7491A) with BamHI and Smal, and was followed by the extremities. were trimmed, and the temperature-sensitive origin of replication was inserted into a pBS4S trimmed-end Ndel site, competent Escherichia coli JM109 cells (Takara Bio Inc.) were used for transformation with that DMA, and the transformed cells were applied to one. LB medium containing 25 pg / ml Km followed by overnight culture Subsequently, the appearing colonies were harvested and single colonies were isolated, and thus transformants were obtained.Plasmids were extracted from transformants and one plasmid. into which a PCR product of interest was inserted was called pBSST Figure 3 shows the procedures for the construction of pBSST.

Example 2 CConstruction of broken Idh gene line> (A) Cloning a fragment to disrupt the lactate dehydrqenase gene A fragment of a lactate dehydrogenase gene (hereinafter abbreviated as ldhj gene derived from BreviJbacterium lactofermentum 2256 whose ORF was The suppressed protein was obtained by cross-PCR using as primers synthetic DNAs designed based on the nucleotide sequence (SEQ ID NO: 37) of the Corynebacterium glutamicum gene ATCC13032 (GenBank Database Accession No. NC_003450), which has already been described. That is, PCR was conducted by a conventional procedure using a BreviJbacterium lactofermentum 2256 chromosomal DNA as tempiate and synthetic DNAs of SEQ ID Nos: 7 and 8 as primers, and thus the extended product of the N-terminal region of the Idh gene. On the other hand, to obtain the expanded product of the C-terminal region of the Idh gene, PCR was performed by a conventional procedure using Brevibacterium lactofermentum 2256 genomic DNA as template and synthetic DNAs of SEQ ID Nos: 9 and 10 as primers. SEQ ID Nos: Θ and 9 are complementary to each other and have structures to suppress all ldh ORF sequences. BrBvibacterium lactofermentum strain 2256 is available from the American Type Culture Collection (ATCC) (Address: ATCC, PO, Box 1549, Manassas, VA 20108, United States of America), then to obtain a fragment of the ldh gene. whose internal sequence is suppressed, the above-mentioned ldh N-terminal and C-terminal gene products were mixed at approximately equimolar concentration and PCR was performed by a conventional procedure using the mixture of genetic products as templates and Synthetic DNAs of SEQ ID Nos: 11 and 12 as primers, thereby producing the amplified ldh gene product introduced by mutation The PCR product obtained in this manner was purified by a standard procedure and then digested with SalI, followed by insertion. at the SBS site of pBS4S described above. Competent cells of Escheríchia coli JM109 (Takara Bio Inc.) were used for transformation with this DNA and the T cells. The formed samples were applied to an LB medium containing 100 µg IPTG, 40 pg X-Gal and 25 µg Km, followed by overnight culture. Subsequently, the white colonies that appeared were harvested and the simple colonies were isolated, thus obtaining transformants. Plasmids were extracted from the transformants and a plasmid into which a PCR product of interest was inserted was called âldh56-1. Figure 4 shows the procedures for plasmid construction. (B) Preparation of broken ldh strain The Aldh56-1 obtained in (A) above does not contain a region that enables autonomous replication in a cell of a coryneform bacterium. Therefore, when a coryneform bacterium is transformed with this plasmid, a strain where the plasmid is integrated into a chromosome by homologous recombination appears with a very low transformant frequency. Brevifoacteriam lactofermentum strain 2256 was transformed using a high concentration of the plasmid óldh56-l by the electric pulse method, and the transformed cells were applied to CM-Dex medium (5 g / l glucose, 10 g / l polypeptone). 0.10 g / l yeast extract, 1 g / l KH2PO4, 0.4 g / l MgS047H20, 0.01 g / l FeS047H20, 0.01 g / l MnS047H20, 3 g / l urea 1.2 g / l soy hydrolyzate, pH 7.5 (KOH)) containing 25 μg kanamycin, followed by cultivation at 31.5 ° C for about 30 hours. A strain grown in this medium contains the kanamycin resistance gene and the sacB gene, which are derived from the plasmid in the genome as a result of homologous recombination between the plasmid ldh gene fragment and the ldh gene in a genome. Brevibacterium lactofermentum strain 2256. The single cross-recombinant was then cultured at 31 ° C overnight in liquid CM-Dex medium containing no kanamycin and, after appropriate dilution, was applied to Dex-SlO medium containing 10% sucrose (100 g / l sucrose, 10 g / l polypeptone, 10 g / l yeast extract, 1 g / l KH2PO4, 0.4 g / l MgS047H20, 0.01 g / l FeS047H20, 0.01 g / l MnS047H20, 3 g / l urea, 1.2 g / l soy hydrolyzate, 10 μg / l biotin, pH 7.5 (KOH) not containing kanamycin, followed by 31.5 ° C for about 30 hours. As a result, about "50 strains were obtained, which were considered to have become sucrose insensitive due to the elimination of the sacB gene by the second homologous recombination.

The strains thus obtained include: a strain in which the Idh gene has been replaced by the mutant type derived from padh56-1, and a strain in which the ldh gene has reverted to the wild type. One can easily verify whether the ldh gene is mutant or wild type by directly submitting the bacterial strains obtained by culture on Dex-SIQ agar to PCR and detecting its ldh gene. In the PCR analysis performed using primers (SEQ ID NOS: 7 and 10) to enlarge the ldh gene, a strain that produced a PCR product smaller than that obtained by PCR performed using a chromosomal DNA. The 2256 lineage as a template was defined as a broken ldh lineage and used in the experiments below. As a result of the analysis of sucrose-insensitive strains by the method mentioned above, a strain carrying only the mutant type gene was selected and named strain 2256Δ (ldh). In addition, the strain was used as the parent strain of the following broken acetic acid biosynthetic gene strain.

Example 3 CConstruction of the broken acetate kinase gene line (A) Cloning a fragment to break the acetate kinase gene A fragment of an acetate kinase gene (hereinafter abbreviated as ack) of the 2256 Brevibacteriuin lactofermentum strain whose ORF was deleted. obtained by cross-PCR using synthetic DNAs designed based on the nucleotide sequence (1945-3135 in SEQ ID NO: 39} of the Corynebacterium glutamicum gene ATCC13032 (GenBank Database Accession Number NC_003450) as primers. That is, PCR was conducted using Brevibacterium lactofermentam strain 2256 genomic DNA as a template and synthetic DNAs of SEQ ID NOS: 13 and 14 as primers, thereby obtaining an extended N-terminal region product. On the other hand, to obtain the extended product of the ack gene · C-terminal region, PCR was performed using a Brevibac teri a lactofermentum 2256 genomic DNA as template and synthetic DNAs of SEQ ID NOS: 15 and 16 as primers. SEQ ID NOS: 14 and 15 are complementary to each other. PCR was conducted using KOD-plus (TOYOBO) such that a thermal retention cycle at 94 ° C for 2 minutes was performed and then, for the N-terminal region, a denaturation cycle at 94 ° C for 10 seconds. annealing at 55 ° C for 30 seconds and stretching at 68 ° C for 30 seconds and, for the C-terminal region, a denaturation cycle at 94 ° C for 10 seconds, annealing at 55 * C for 30 seconds and stretching at 68 eC for 2 minutes were repeated 30 times, respectively. Then, to obtain a fragment of the ack gene whose internal sequence is suppressed, the aforementioned genetic products from the ack N-terminal and C-terminal regions were mixed at approximately equimolar concentration and PCR was performed using the mixing the genetic products as templates and synthetic DNAs of SEQ ID NOS: 17 and 18 as primers, thereby producing the extended mutated ack gene product. PCR was performed using KOD-plus (TOYOBO) so that a thermal retention cycle of 94 ° C was performed for 2 minutes and then a denaturation cycle at 94 ° C for 10 seconds, annealing at 55 °. C for 30 seconds and stretching at 68 ° C for 2.5 minutes was repeated 30 times, thereby producing an extended product of the mutated introduced ack gene of interest. The PCR product obtained in this manner was purified by a standard procedure and then digested with XbaI, followed by insertion into the XbaI site in pBSST constructed in Example 1 (C) above. Competent Escherichia coli JM109 cells (Takara Bio Inc.) were used for transformation with this DNft and transformed cells were applied to an LB medium containing 100 μΜ IPTG, 40 Mg / ml X-Gal and 25 pg / ml kanamycin , followed by culture overnight. Subsequently, the appearing white colonies were harvested, and single colonies were isolated, and thus transformants were obtained. Plasmids were extracted from the transformants and a plasmid into which a PCR product of interest was inserted was called pBSST :: âack. Figure 5 shows the procedures for building pBSST :: üack. <B3 Preparation of broken ack strain The origin of replication for coryneform bacteria in pBSST :: ack obtained in (A) above is temperature sensitive. That is, the plasmid may replicate autonomously in a cell of a coryneform bacterium at 25 ° C, but does not replicate autonomously at 31.5 * 0 (or 34 ° C). Brevibacteri an lactofermentum strain 2256ώ (Idh) was transformed by the electrical pulse method using the plasmid, and the transformed cells were applied to a CM-Dex medium containing 25 µg / ml kanamycin, followed by culture at 25 ° C. for two nights. The colonies that appeared were isolated, thus producing transformants. Transformants contain the plasmid. Transformants were grown at 34 ° C overnight in CM-Dex medium (5 g / l glucose, 10 g / l polypeptone, 10 g / l yeast extract, 1 g / l KH2P04, 0.4 g / l MgS047H2O, 0.01 g / l FeS047H20, 0.01 g / l MnS047H20, 3 g / l urea, 1.2 g / l soy hydrolyzate, pH 7.5 { KOH}) not containing kanamycin and, after appropriate dilution, were applied to a CM-Dex medium containing 25 Mg / ml kanamycin, followed by culturing at 34 ° C for about 30 hours. kanamycin resistant gene and the sacB gene, which are derived from the plasmid in the genome, as a result of the homologous recombination between the plasmid ack gene fragment and the Brevibacterium lactofermentam 2256Δ (Idh) genome.

The single cross-recombinant was then grown at 31.5 ° C overnight in CM-Dex liquid medium containing no kanamycin and, after appropriate dilution, was applied to Dex-SlO medium containing 101 sucrose and not containing kanamycin. followed by culture at 31.5 ° C for about 30 hours. As a result, about 50 strains were obtained which were considered to have become sucrose-insensitive due to the elimination of the sacB gene by the second homologous recombination.

The lines thus obtained include; a strain in which the ack gene was replaced by the mutant type derived from pBSST :: üack, and a strain in which the ack gene reverted to the wild type. Whether the ack gene is mutant or wild type can be easily ascertained by directly subjecting the bacterial strain obtained by culture on Dex-SlO agar medium to PCR and detecting the ack gene. Analysis of the ack gene using primers (SEQ ID NOS: 13 and 16} for PCR amplification should result in a 3.7 kb wild type DMA fragment and a 2.5 kb DMA fragment for the PCR. mutant type having a suppressed region.As a result of the analysis of the sucrose-insensitive strain by the method mentioned above, a strain carrying only the mutant type gene was selected and named 2256Δ (Idh, ack).

Example 4 cConstruction of a line in which the acetate kinase gene and the phosphotransacetylase gene are disrupted> (A) Cloning fragments to disrupt the acetate kinase gene and the phosphotransacetylase gene The acetate kinase r'ack "ORFs) and Phosphotransacetylase gene (hereinafter referred to as pta) of Brevibacterium lactofermentam strain 2256 have an operon structure, and both ORFs can become simultaneously deficient.These gene fragments were obtained by cross-PCR using synthetic DNAs designed based on the sequences of nucleotides of the Corynebacterium glutamicum ATCC13032 genes (GenBank Database Accession No. NC_003450) (pta-ack gene; SEQ ID NO; 39) That is, PCR was conducted using a genomic DNA from the 2256 strain of Brevibacteri is a lactoferment as a template, and the synthetic DNAs of SEQ ID NOS: 19 and 20 as primers, thereby producing an extended product of the N-terminal region of the pta gene. On the other hand, to produce an extended C-terminal region product of the ack gene, PCR was performed using a Brevxbacterium 1actofermentum 2256 genomic DNA as template, and synthetic DNAs of SEQ ID NOS; 21 and 16 as primers. SEQ ID NOS: 20 and 21 are partially complementary to each other. PCR was performed using KOD-plus (TOYOBO) so that a thermal retention cycle at 94 ° C was performed and then, for the N-terminal region, a denaturation cycle at 94 ° C for 10 seconds, annealing at S5 ° C for 30 seconds and stretching at 68 ° C for 30 seconds and, for the C-terminal region, a denaturing cycle at 94 ° C for 10 seconds, annealing at 55 * C for 30 seconds and stretching at 68 ° C. "C for 2 minutes were repeated 30 times, respectively. Then, to obtain a fragment of the pta-ack gene in which an internal sequence of pta and ack is deleted, the above-mentioned N-terminal region genetic products of pta and ack C-terminal region were mixed at approximately equimolar concentration, and PCR was performed using the mixture as templates and synthetic DNAs of SEQ ID NOS: 22 and 18 as primers to thereby produce the extended product. of a mutated pta-ack gene, PCR was performed using ndo-ee- KOD-p (TOYOBO) such that a thermal retention cycle was performed at 94 ° C for 2 minutes and then a denaturation cycle at 94 ° C for 10 seconds, annealing at 55 ° C for 30 seconds, and elongation at 68 ° C for 2.5 minutes was repeated 30 times, thereby producing an extended product of the mutated pta-ack gene of interest. The PCR product obtained in this manner was purified by a standard procedure and then digested with XbaI, followed by insertion into the pBS5T XbaI site. Competent Escherichia co JM109 cells (Takara Bío Inc.) were used for transformation with this DMA and the transformed cells were applied to an LB medium containing 100 μ IPTG, 40 μg / ml X-Gal and 25 μ9 / ιη1 kanamycin, followed by overnight culture. Subsequently, the white colonies that appeared were harvested and the simple colonies were isolated and thus transformants were obtained. Plasmids were extracted from the transformants and a plasmid into which a PCR product of interest was inserted was called pBSST :: apta-ack. Figure 6 shows the procedures for building pBSST :: ôpta-ack. (B) Preparation of broken pta-ack strain The origin of replication for coryneform bacteria in pBSST :: ôpta-ack obtained by procedure (A) above is temperature sensitive. That is, the plasmid is autonomously replicable in a cell of a coryneform bacterium at 25 ° C, but is not autonomously replicable at 31.5 ° C (or 34 ° C). Brevibacterium lactofermentum strain 2256Δ (ldh) was transformed using the plasmid by the electric pulse method, and the transformed cells were applied to a CM-Dex medium containing 25 μς / ιηΐ of kanamycin, followed by culture at 25 ° C by 2 nights The colonies that appeared were isolated, producing transformants * Transformants have the plasmid, ........... Transformants were grown at 34 ° C overnight in a CM-Dex liquid medium not containing kanamycin and , after appropriate dilution, were applied to a CM-Dex liquid mix containing 25 μς / ml kanamycin, followed by culturing at 34 ° C for about 30 hours.The medium grown strain contains the kanamycin resistance gene and sacB gene derived from the plasmid in the genome as a result of homologous recombination between the pta-ack gene fragment in the plasmid and the pta-ack gene in the Brevibacterium lactofermentum 2256Δ (Idh) genome.

In. The single crossover recombinant was then cultured at 31.5 ° C overnight in CM-Dex liquid medium not containing kanamycin and, after appropriate dilution, was applied to Dex-SlO medium containing 10 'sac rose and not containing kanamycin, followed by culture at 31.5 ° C for about 30 hours. As a result, about 50 strains were obtained, which were considered to have become insensitive to sac rose due to the elimination of the sacB gene by the second homologous recombination.

The strains obtained in this manner include: a strain in which the pta and ack genes have been replaced by the pBSBT :: Apta-ack-derived mutant type, and a strain in which the pta and ack genes have been reverted to the wild type. It is easy to verify whether the pta and ack genes are mutant or wild type genes by directly PCRing a bacterial strain obtained from culture on a Dex-SlO agar medium and detecting the pta and ac genes. ack Analysis of the pta-ack gene using primers (SEQ 1D NOS: 19 and 16) for PCR amplification should result in a 5.0 kb wild type DNA fragment and a 2.7 kb DNA fragment for PCR. the mutant type having a suppressed region.

As a result of the analysis of the sucrose-insensitive strain by the above method, a strain carrying only the mutant type gene was selected and named 2256A (ldh, pta, ack).

Example 5 cBuilding of a disrupted pyruvate oxldase gene line (A) Cloning a fragment to disrupt the pyruvate oxidase gene A pyruvate oxidase (hereinafter abbreviated to poxB) gene fragment of the Brevibacterium lactofezmentum 2256 strain whose ORF was deleted by PCR using synthetic DNAs designed based on the nucleotide sequence (SEQ ID NO: 42) of the Corynebacterium glutamicum ATCC13032 gene (GenBank Database Accession No. NC_003450) as described above. That is, PCR was performed using Brevibacterium lactofermentum strain 2256 genomic DNA as tempiate and synthetic DNAs of SEQ ID NOS: 23 and 24 as primers, thereby obtaining an extended product of the N-terminal region of the gene. from poxB.

On the other hand, to obtain the extended product of the C-terminal region of the poxB gene, PCR was performed using a genomic DNA of Brevibacterí 22 lactofermentum as a template and synthetic DNAs of SEQ ID NOS: 25 and 26 as primers. SEQ ID NOS: 24 and Ξ5 are complementary to each other. The PCR was conducted using KOD-plus (TOYOBO) such that a heat retention cycle was performed at 94 ° C for 2 minutes and then a denaturation cycle at 94ec for 10 seconds, annealing at 55 °. C for 30 seconds and stretching at 68 ° C for 40 seconds was repeated 30 times for both N-terminal and C-terminal regions. Then, to obtain a poxB gene fragment whose internal sequence is suppressed, the above-mentioned poxB N-terminal and C-terminal gene products were mixed at approximately equiroolar concentration and PCR was performed using mixes as template and synthetic DNAs of SEQ XD NOS: 27 and 28 as primers, thereby producing the extended product of a mutated poxB gene. PCR was conducted using KOD-plus (TOYOBO) such that a thermal retention cycle was performed at 94 ° C for 2 minutes and then a denaturation cycle at 94 ° C for 10 seconds, annealing at 55 ° C. for 30 seconds and elongation at 68 ° C for 70 seconds was repeated 30 times, thereby producing an extended product of the mutated poxB gene of interest. The PCR product thus obtained was purified by a standard procedure and then digested with XbaI, followed by insertion into the XbaI site of pBS5T constructed in Example 1 (C) as described above. Competent Escherichia coli JM1Q9 cells (Takara Bio Inc.) were used for transformation with this DNA and the transformed cells were applied to an LB medium containing 100 μΜ XPTG, 40 μ9 / πι1 X-Gal, and 25 pg / ml of kanamycin, followed by overnight culture. Subsequently, the white colonies that appeared were harvested and the simple colonies were isolated and thus transformants were obtained. Plasmids were extracted from the transformants and a plasmid into which a PCR product of interest was inserted was called pBSSTr: ΔροχΒ. Figure 7 shows the procedures for building pBSST :: ΔροχΒ. (B) Preparation of broken poxB strain The origin of replication for coryneform bacteria in pBS5T :: ΔροχΒ obtained in Example (A) described above is temperature sensitive. That is, the plasmid is autonomously replicable in a cell of a coryneform bacterium at 25 eC, but is not autonomously replicable at 31.5 ° C (or 34 * C). The Brêvibacterium lactofexmenLvm 2256Δ (ldh, pta, ack) strain was transformed using the plasmid by the electric pulse method, and the transformed cells were applied to a CM-Dex medium containing 25 μς / ιηΐ of kanamycin, followed by culture at 55 ° C for 2 nights. The colonies that appeared were isolated, producing transformants. Transformants must have the plasmid.

Transformants were cultured at 34 ° C overnight in a CM-Dex liquid medium not containing kanamycin and, after appropriate dilution, were applied to a CM-Dex liquid medium containing 25 μ9 / ιη1 of kanamycin, followed by 34 ° C for about 30 hours. In a medium grown medium, the kanamycin resistance gene and the plasmid-derived sacB gene are inserted into the genome as a result of homologous recombination between the poxB gene fragment in the plasmid and the poxB gene in the genome. Brevibac strain 2256Λ (ldh, pta, ack) have lactofermentum fight. The single crossover recombinant was then cultured at 31.5 ° C overnight in CM-Dex non-kanamycin-containing liquid medium and, after appropriate dilution, was applied to Dex-SlO medium containing 10% sucrose and not containing kanamycin, followed by culturing at 31.5 ° C for about 30 hours. As a result, about 50 strains were obtained, which were considered to have become sucrose-insensitive due to the elimination of the sacB gene by the second homologous recombination. The strains obtained include: a strain in which the poxB gene has been replaced by the mutant type. derived from pBS5T :: ΔροχΒ, and a strain in which the poxB gene reverted to wild type. Whether the pcxB gene is mutant or wild type can be readily ascertained by subtracting a bacterial lineage obtained by culturing on a Dex-SlO agar medium directly to PCR and detecting the poxB gene. 2.4 kg wild type DMA fragment and a 1.2 kb mutant type DNA fragment having the suppressed region can be detected by poxB gene analysis with primers (SEQ ID NOS: 23 and 26} As a result of the analysis of the sucrose-insensitive strain by the method mentioned above, a strain carrying only the mutant type gene was selected and named 2256 (ldh, pta, ack, poxB).

Example 6 cBuilded Aeetyl-CoA Hydrolase Gene Lineage Construction> (A) Cloning a Fragment to Disrupt the Acetyl-CoA Hydrolase Gene An Acetyl-CoA Hydrolase gene fragment (hereinafter abbreviated as ach) of the 2256 Brevibacterium strain suppressed lactoferiuentum was obtained by cross-PCR using synthetic DNAs designed based on the nucleotide sequence (SEQ ID NO: 44) of the Corynebacterium glutamicum ATCC13032 gene (GenBank Database Accession No. NCJ303450), that has already been described. That is, PCR was performed using a Brevibacterium lactofermentum strain 2256 genomic DMA as tempiate and synthetic DNAs of SEQ ID NQS: 29 and 30 as primers, thereby obtaining an extended product of the C-terminal region of the gene. from ach. On the other hand, to obtain the extended product of the ach gene N-terminal region, PCR was performed using a genomic DNA from Brevibacterium lactofermentum 2256 as the template and synthetic DNAs of SEQ ID NOS: 31 and 32 as pri / ners. SEQ ID NOS: 30 and 31 are complementary to each other. PCR was conducted using KOD-plus (TOYOBO) such that a heat retention cycle was performed at 94 ° C for 2 minutes and then a denaturation cycle at 94 ° C for 10 seconds, annealing at 100 ° C. 55Â ° C for 30 seconds and stretching at 68Â ° C for 50 seconds was repeated 30 times for the N-terminal region and C-terminal region. Next, to obtain a fragment of the ach gene in which an internal sequence is suppressed, the above-mentioned ach N-terminal and C-terminal gene products were mixed at approximately equimolar concentration, and FCR was performed using the mixture as template and synthetic DNAs of SEQ ID NOS: 33 and 34 as primers, thereby producing the extended product of a mutation-introduced ach gene. PCR was performed using KOD-plus (TOYOBO) such that a thermal retention cycle was performed at 94 ° C for 2 minutes and then a denaturation cycle at 94 ° C for 10 seconds, annealing at 55 ° C for 30 seconds and elongation at 68 ° C for 90 seconds was repeated 30 times, thereby producing an enlarged ach gene product of interest introduced by mutation. The PCR product thus obtained was purified by a standard procedure and digested with XbaI, followed by insertion into the XbaI site of pBS4S constructed in Example 1 (B) as described above. Competent cells of Escherichia coli JM109 (Takara Bio Inc.) were used for transformation with this DNA and the transformed cells were applied to an LB medium containing 100 μΜ IPTG, 40 pg / ml X-Gal, and 25 μς / ml kanamycin, followed by overnight culture. Subsequently, the white colonies that appeared were harvested and the simple colonies were isolated, and thus transformants were obtained. The plasmids were extracted from the transformants and a plasmid into which a PCR product of interest was inserted was called pBS4S ;; ôach. Figure 8 shows the procedures for building pBS4S :: ôach. (B) Preparation of broken ach line The pBS4S :: Aach obtained in (A) above does not include a region that enables autonomous replication in a cell of a coryneform bacterium, so when a coryneform bacterium is transformed with the plasmid, a strain wherein the plasmid is integrated into a chromosome by homologous recombination appears at a very low frequency as a transformant. The 2256A (ldh) strain, 2256A (ldh, pta, ack) strain, and the Brevibacterium lactofermentam 2256A (ldh, pta, ack, poxB) strain were transformed using a high concentration of plasmid pBS4S :: üach by the method pulses and transformed cells were applied to CM-Dex medium containing 25 pg / ml kanamycin, followed by culturing at 31.5 ° C for about 30 hours. In the growing medium, the kanamycin resistance gene and plasmid-derived sacB gene are inserted into the genome as a result of homologous recombination between the ach gene fragment in the plasmid and the ach gene in each genome. from the 2256Δ (ldh), 2256Δ (ldhf pta, ack), 2256Δ (ldh, pta, ack, poxB) and 2256Δ (ldh, pta, ack, poxB, acp) strains of lactofermentam.

The single cross-recombinant was then grown at 31.5 ° C overnight in liquid CM-Dex medium containing no kanamycin and, after appropriate dilution, was applied to Dex-SlO medium containing 10% sucrose and not containing kanamycin. followed by culture at 31.5 ° C for about 30 hours. As a result, about 50 strains were obtained, which were considered to have become sucrose insensitive due to the elimination of the sacB gene by the second homologous recombination.

The strains thus obtained include: a strain in which the ach gene has been replaced by the mutant type derived from pBS4S :: & ach, and a strain in which the ach gene has reverted to wild type. Whether the ach gene is mutant or wild type can be easily verified by subjecting a bacterial strain obtained by culturing it on a Dex-SlO agar medium directly to PCR and detecting the ach gene. Analysis of the ach gene using prime rs (SEQ ID NOS: 29 and 32) for PCR amplification should result in a 2.9 kb wild type ONA fragment and a 1.4 kb DNA fragment for the mutant type that has a suppressed region. As a result of the analysis of the sucrose-insensitive strain by the above method, a strain carrying only the mutant type gene was selected and strains obtained from 2256â (ldh), 2256A (ldh, pta, ack), 2256A (ldh, pta, ack, poxB) and 2256Δ (ldh, pta, ack, poxB, acp) were named 2256Δ (ldh, ach), 2256A (ldh, pta, ack, ach) and 2256A (ldh, pta, ack, poxB, ach) ) respectively.

Example 7 <Production of succinic acid by the broken ach strain> (A) Culture evaluation of the ach deficient strain The 2256Δ (ldh) strain and the 2256Δ (ldh, ach) strain from Brevibacterium lactofermentum were used to produce succinic acid as follows. . The 2256Δ (ldh) and 2256Δ (ldh, ach) bacterial cells obtained by culturing in a CM-Dex plated medium were inoculated into 3 ml of seeded culture medium (10 g / l glucose, 2.5 g / l (NH4) 2 SO4, 0.5 g / l KH2 PO4, 0.25 g / l MgS0 7H20, 2 g / l urea, 0.01 g / l FeSG4 7Β20, 0.01 g / L MnSCU 7H20, 50 pg / l biotin, 100 pg / l VB1-HCl, 15 mg / l protocatechuic acid, 0.02 mg / l CuSO4, and 10 mg / l CaCl2, pH 7 .0 (KOH)). The culture was performed with shaking in a test tube at 31.5 ° C for about 15 hours under aerobic condition.

Thereafter, 3 ml of a main medium (70 g / l glucose, 5 g / l (NH4) 2 SO4, 2 g / l KH2PO4, 3 g / l urea, 0.01 g / l) were added to the tube. L FeS04 7H20, 0.01 g / L MnSG4 7H20, 200 pg / L Biotin, 200 pg / L VBl-HCl, 40 g / L MOPS, 50 g / L MgCOj, pH 6.8 (NaOH)). To prevent aeration, the culture for succinic acid production was performed with the hermetically sealed tube with a silicone cap. The culture was performed with stirring at 31.5 ° C for about 24 hours and was terminated before the sugar contained in the kinda ran out.

Upon completion of the culture, the accumulated amounts of succinic acid and acetic acid by-product in the medium were analyzed by liquid chromatography after the culture liquid had been adequately diluted. A column obtained by connecting two pieces of Shim-pack SCR-102H (Shimadzu) in series was used and the sample was eluted at 40 ° C using 5 mM p-toluene sulfonic acid, the eluent was neutralized using 20 mM Bis-Tris aqueous solution containing 5 mM p-toluene sulphonic acid and 100 μΜ i EDTA. Succinic acid and acetic acid were measured by determining the electrical conductivity by CDD-1GAD (Shimadzu). The results are shown in Table 1.

The yield of the 2256Δ (ldh, ach) strain was found to have improved by 6% compared to the 2256Δ (ldh) strain as the parent strain.

Table 1, Production of succinic acid and acetic acid in disrupted Brevibacterium lactofermentum strains 2256Δ (ldh) and 2256Δ (ldh, ach, pta, ack) were used in culture to produce succinic acid as follows. Bacterial cells ..... from strain 2256á (ldh) and strain 2256A (ldh, ach, pta, ack) obtained by culturing in a plated Cm-Dex medium were inoculated into 3 ml of the seeded culture medium mentioned above, The culture was shaken in a test tube at 31.5Â ° C for about 15 hours under aerobic condition.

Thereafter, 3 ml of the above-mentioned main medium was added to the test tube. To prevent aeration, the culture for succinic acid production was conducted with the hermetically sealed tube with a silicone cap. The culture was performed with stirring at 31.5 ° C for about 24 hours and was terminated before the sugar contained in the medium was depleted.

Upon completion of culture, the amounts of succinic acid and acetic acid by-product accumulated in the medium were analyzed by liquid chromatography after the culture liquid had been properly diluted. A column obtained by connecting two pieces of Shim-pack was used. SCR-1Q2H (Shimadzu) serially and the sample was eluted at 4 ° C using 5 mM p-toluene sulfonic acid. The eluent was neutralized using 20 mM Bis-Tris aqueous solution containing 5 mM p-toluene sulfonic acid and 100 μΜ EDTA. Succinic acid and acetic acid were measured by determining electrical conductivity using CDD-10AD (Shimadzu). The results obtained are shown in Table 2. In the case of the 2256Δ strain (ldh, ach, pta, ack), the succinic acid production level was equal to that of the 2256i (ldh) parent line, but the acetic acid level in relation to succinic acid was reduced to about one third of the 2256Δ (ldh, ach) strain level and to about half of the 2256Δ (ldh) strain level, which showed that the production of acetic acid as a by-product decreased dramatically. The result and result described in (A) above indicated that both elimination and decrease of either PTA-ACK activity or ACH activity are ineffective in reducing acetic acid, but acetic acid is dramatically reduced by eliminating or diminishing all these activities. At the same time, it is easily assumed that eliminating or decreasing PTA or ACK activity along with ACH activity is effective for reducing acetic acid. As for succinic acid production, it has been found that eliminating or reducing only ACH activity is effective.

Table 2 - Production of broken succinic acid and acetic acid in poxB The 2256Δ (Idh) strain, the 2256Δ (ldh, pta-ack, ach) strain, and the Brevibacterium 2256Δ (ldh, pta-ack, poxB, ach) strain lactofermentum were used in culture for the production of succinic acid as follows. Bacterial cells from strain 2256Δ (ldh), strain 2256Δ (ldh, pta-ack, ach), and strain 2256Δ (ldh, pta-ack, poxB, ach) cultured in a plated Cm-Dex medium were inoculated. in 3 ml of the seeded culture medium mentioned above. The culture was shaken in a test tube at 31.5 ° C for about 15 hours under aerobic condition.

Thereafter, 3 ml of the main medium mentioned above was added to the test tube. To prevent aeration, the culture for succinic acid production was conducted with the hermetically sealed tube with a silicone cap. Cultivation was performed with stirring at 31-5 ° C for about 24 hours and was terminated before the sugar contained in the medium was depleted. Upon completion of the culture, the amounts of succinic acid and acetic acid by-product accumulated in the medium were analyzed by liquid chromatography after the culture liquid had been adequately diluted. A column obtained by connecting two pieces of Shim-pack SCR-102H (Shimadzu) in series was used and the sample was eluted at 40 ° C using 5 mM of 1 p-toluene sulfonic acid. The eluent was neutralized using 20 mM Bis-Tris aqueous solution containing 5 mM p-toluene sulfonic acid and 100 μΜ EDTA. Succinic acid and acetic acid were measured by determining the electrical conductivity by CDD-10AD (Shimadzu). The results obtained are shown in Table 3.

In the case of lineage 2256Δ (ldh, pta, ack, ach, poxB) prepared with the additional disruption of poxB in lineage 2256Δ (ldh, pta, ack, ach), acetic acid further decreased by about 40% compared to parent line 2256Δ (ldh, pta, ack, ach). The result revealed that eliminating or decreasing ach activity along with any or all of pta, ack and poxB activities is effective for reducing acetic acid.

Table 3. Production of succinic acid and acetic acid in the use of the bacteria of the present invention enables efficient production of succinic acid. Succinic acid is useful as a feedstock for a biodegradable polymer and the like.

Claims

Claims (7)

1. Method for the production of succinic acid, characterized by; understand the steps of: allowing a coryneform bacteria selected from the group consisting of Brevibacterrum lactofermentum and Corynebecterium glutamicura having an ability to produce succinic acid to act on an organic feedstock in a reaction liquid containing a carbonate ion, a bicarbonate ion or carbon dioxide for generating and accumulating succinic acid in the reaction liquid and collecting succinic acid from the reaction liquid, said bacterium having been modified such that an acetyl-CoA hydrolase activity having an amino acid sequence From SEQ ID NO: 45 is decreased by disruption of an acetyl CoA hydrolase gene on a chromosome. Method according to Claim 1, characterized in that the acetyl CoA hydrolase gene is a DNA comprising one. nucleotide sequence numbered 1037 to 2542 in SEQ ID NO: 44. A method according to claim 1 or 2, characterized in that said bacterium has been further modified such that the activities of one or both phosphotransacetylase and acetate kinase are decreased by interruption of a phosphotransacetylase gene comprising a sequence. of nucleotide numbers 956 to 194 2 in SEQ ID NO: 39 and / or an acetate kinase gene comprising * a nucleotide sequence numbered from 1945 to 3135 in SEQ ID NO: 39 of a chromosome. Method according to any one of claims 1 to 3, characterized in that said bacterium has been further modified such that a lactate dehydrogenase activity is decreased by disruption of a lactate dehydrogenase gene comprising a nucleotide sequence. blinks numbers from 898 to 1854 in 5EQ ID NO: 37 of a chromosome. Method according to any one of claims 1 to 4, characterized in that said bacterium has been further modified such that a pyruvate carboxylase activity is increased by transformation with a vector containing the pyruvate carboxylase gene comprising a sequence. of nucleotides represented by SEQ XD NO: 46 6. Method according to any one of claims 1 to 5, characterized in that; The coryneform bacterium or its treated product may be allowed to act on the organic raw material under anaerobic conditions. A method for the preparation of a succinic acid-containing polymer, characterized in that it comprises the succinic acid production steps according to any one of claims 1 to 6 and additionally a succinic acid polymerization step obtained.