JP5663859B2 - Non-amino organic acid producing bacteria and method for producing non-amino organic acid - Google Patents

Description

The present invention relates to a novel non-amino organic acid-producing bacterium and production of a non-amino organic acid using the same.

When producing non-amino organic acids such as succinic acid by fermentation, anaerobic bacteria such as Anaerobiospirillum genus and Actinobacillus genus are usually used (Patent Documents 1 and 2, Non-Patent Document 1). When anaerobic bacteria are used, the yield of the product is high, but on the other hand, a large amount of organic nitrogen sources such as CSL (corn steep liquor) in the medium because it requires many nutrients to grow. Need to be added. Adding a large amount of these organic nitrogen sources not only leads to an increase in culture medium cost, but also increases the purification cost when taking out the product, which is not economical.

In addition, aerobic bacteria such as coryneform bacteria are once cultured under aerobic conditions, and after growing the cells, they are collected and washed, and non-amino organic acids are used as stationary cells without aeration of oxygen. A production method is also known (Patent Documents 3 and 4). In this case, it is economical because the amount of organic nitrogen added is small and sufficient growth can be achieved using a simple medium. However, the amount of non-amino organic acid produced, the concentration, and the amount per cell There was room for improvement, such as improvement in production speed and simplification of the manufacturing process.

As a method for fermentative production of non-amino organic acids using recombinant microorganisms, a method using coryneform bacteria in which the activity of phosphoenolpyruvate carboxylase is enhanced has been reported (for example, see Patent Document 5). The production of non-amino organic acids using bacteria with enhanced activity of sugar phosphotransferase (PTS), which is involved in the uptake of NO, has not been reported so far. Non-Patent Document 2 discloses a ptsS gene-disrupted strain, which suggests that ptsS is involved in sucrose uptake, but does not disclose a strain with enhanced ptsS gene expression. The relationship between ptsS and the production of non-amino organic acids such as succinic acid was unclear.

US Pat. No. 5,143,834 US Pat. No. 5,504,004 JP-A-11-113588 JP 11-196888 A Japanese Patent Laid-Open No. 11-196887

International Journal of Systematic Bacteriology (1999), 49,207-216 FEMS Microbiol Lett. 2005 Mar 15; 244 (2): 259-66.

The subject of this invention is providing the manufacturing method of non-amino organic acid with higher production efficiency.

As a result of intensive studies to solve the above problems, the present inventor obtained a bacterium or a treated product thereof modified so that the expression of the ptsS gene is enhanced as compared with the unmodified strain. It was found that the yield of non-amino organic acids such as succinic acid per sucrose consumed per sucrose was increased by acting on sucrose in a reaction solution containing ions or carbon dioxide gas, and the present invention was completed.

That is, according to the present invention, the following inventions are provided.
(1) A bacterium that has a non-amino organic acid-producing ability and has been modified so that expression of the ptsS gene is enhanced as compared to a non-modified strain.
(2) The bacterium according to (1), wherein the ptsS gene has a homology of 80% or more with the amino acid sequence of SEQ ID NO: 10 and encodes a protein having PtsS activity.
(3) The bacterium according to (1) or (2), which is further modified so that lactate dehydrogenase activity is reduced as compared with an unmodified strain.
(4) The bacterium according to any one of (1) to (3), further modified so that pyruvate carboxylase activity is enhanced as compared with an unmodified strain.
(5) Further, the activity of one or more enzymes selected from acetate kinase, phosphoorance acetylase, pyruvate oxidase and acetyl-CoA hydrolase was modified so as to be reduced as compared with an unmodified strain. The bacterium according to any one of (1) to (4).
(6) The bacterium according to any one of (1) to (5), which is a coryneform bacterium.
(7) The bacterium according to (6), wherein the coryneform bacterium is Corynebacterium glutamicum, Brevibacterium flavum, or Brevibacterium lactofermentum.
(8) In the reaction solution, the bacterium according to any one of (1) to (7) or a processed product thereof is allowed to act on sucrose in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas. A method for producing a non-amino organic acid, comprising producing and accumulating a non-amino organic acid and collecting the non-amino organic acid.
(9) The method according to (8), wherein sucrose is allowed to act in an anaerobic atmosphere.
(10) The method according to (8) or (9), wherein the non-amino organic acid is succinic acid.
(11) A method for producing a succinic acid-containing polymer, comprising a step of producing succinic acid by the method according to (10) and a step of polymerizing succinic acid obtained in the step.
(12) A method for producing a succinic acid derivative, comprising a step of producing succinic acid by the method described in (10), and a step of synthesizing a succinic acid derivative using succinic acid obtained in the step as a raw material.

According to the production method of the present invention, in the production of a non-amino organic acid such as succinic acid using sucrose, the yield of the non-amino organic acid per consumed sucrose is improved and the non-amino organic acid is efficiently produced. Can do.
The obtained non-amino organic acid can be used for food additives, pharmaceuticals, cosmetics and the like. Moreover, a non-amino organic acid containing polymer can also be manufactured by performing a polymerization reaction using the obtained non-amino organic acid as a raw material.

Hereinafter, embodiments of the present invention will be described in detail.
The bacterium of the present invention is a bacterium which has a non-amino organic acid-producing ability and has been modified so that expression of the ptsS gene is enhanced as compared with the unmodified strain.

Here, “having the ability to produce a non-amino organic acid” means that a non-amino organic acid can be produced and accumulated in the medium when the bacterium is cultured in the medium. Non-amino organic acids mean organic acids other than amino acids, but succinic acid, malic acid, fumaric acid, citric acid, isocitric acid, 2-oxoglutaric acid, cis-aconitic acid and pyruvic acid are preferred, and succinic acid, malic acid Dicarboxylic acids such as fumaric acid are more preferred, and succinic acid is particularly preferred.

The ptsS gene in the present invention means a gene encoding a protein having an activity of transferring a phosphate group of phosphoenolpyruvate to sucrose and simultaneously incorporating it into Cytoplasm (EC2.7.1.69: hereinafter referred to as PtsS activity). . PtsS activity can be measured, for example, by the method described in J Bacteriol. 1989 Jan; 171 (1): 263-71.

“Enhanced expression of the ptsS gene” means that the expression level of the ptsS gene is increased as compared to a non-modified strain such as a wild-type strain. The expression level of the ptsS gene is preferably enhanced 1.5 times or more per unit cell weight, and more preferably enhanced 2 times or more, compared to the unmodified strain.

The bacterium of the present invention may be one that has been modified to enhance the expression level of the ptsS gene in a bacterium that originally has a non-amino organic acid-producing ability or a bacterium that has been given a non-amino organic acid-producing ability by breeding. Further, it may be modified so as to have a non-amino organic acid-producing ability by performing a modification that enhances the expression level of the ptsS gene. Examples of means for imparting non-amino organic acid-producing ability by breeding include mutation treatment and gene recombination treatment. For example, when imparting succinic acid-producing ability, lactate dehydrogenase activity as described below is reduced. And other modifications that enhance pyruvate carboxylase activity.

The bacterium used in the present invention can be obtained by using a bacterium as shown below as a parent strain and modifying the parent strain. The kind of the parent strain is not particularly limited, but is preferably a coryneform bacterium, a Bacillus bacterium, or a Rhizobium bacterium, and more preferably a coryneform bacterium.
Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus (Bacillus subtilis), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus subtilis)
pumilus), Bacillus stearothermophilus and the like, and Rhizobium etli and the like as the genus Rhizobium.
Examples of coryneform bacteria include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, or bacteria belonging to the genus Arthrobacter, and among these, those belonging to the genus Corynebacterium or Brevibacterium More preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes or Brevibacterium lactofermentum Bacteria belonging to

Particularly preferred specific examples of the parent strains of the above bacteria include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Coryne And bacteria glutamicum ATCC31831 and Brevibacterium lactofermentum ATCC13869. Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH, International Journal of
Systematic Bacteriology, 1991, vol. 41, p255-260), in the present invention, Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are respectively Corynebacterium glutamicum MJ- It shall be the same strain as 233 strain and MJ-233 AB-41 strain.
Brevibacterium flavum MJ-233 was established on April 28, 1975 by the Ministry of International Trade and Industry, Ministry of International Trade and Industry, Microbial Industrial Technology Research Institute (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (305-8566 Japan) Deposited as FERM P-3068 at Tsukuba City, Ibaraki Prefecture, 1-chome, 1-chome, 1st 6th), transferred to an international deposit based on the Budapest Treaty on May 1, 1981, and deposited with a deposit number of FERM BP-1497 Has been.

The bacterium used as a parent strain in the method of the present invention is not only a wild strain, but also a mutant obtained by normal mutation treatment such as UV irradiation or NTG treatment, genetic techniques such as cell fusion or gene recombination. Any strain such as a recombinant strain to be induced may be used.

The expression level of the ptsS gene can be enhanced by gene recombination, for example, by increasing the copy number of the ptsS gene or by replacing the promoter of this gene.
In the case of modification so that the expression level of the ptsS gene is enhanced, the gene that can be used is not particularly limited as long as it encodes a protein having PtsS activity. For example, Corynebacterium glutamicum having the base sequence of SEQ ID NO: 9 Mention may be made of genes derived from them. In addition, as long as the ptsS gene encodes a protein having PtsS activity, DNA that hybridizes under stringent conditions with DNA having a complementary sequence of the above base sequence, or with the above base sequence, 80% or more, preferably It may be a homologue such as DNA having a homology of 90% or more, more preferably 95% or more, particularly preferably 99% or more. Here, as stringent conditions, it corresponds to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which is a normal washing condition for Southern hybridization. The conditions for hybridizing at a salt concentration are included.
The ptsS gene has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more with the amino acid sequence of SEQ ID NO: 10, and encodes a protein having PtsS activity. It may be DNA.
Further, the ptsS gene may be a DNA encoding a protein having a PtsS activity, having a sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 10. . Here, one or several means preferably means 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5.

Further, coryneform bacteria other than Brevibacterium flavum MJ-233, or ptsS genes derived from other microorganisms or animals and plants can also be used. A ptsS gene derived from a microorganism or animal or plant is isolated from a chromosome of a microorganism, animal or plant, etc., based on a gene whose base sequence has already been determined, or a gene encoding a protein having PtsS activity based on homology with SEQ ID NO: 9, etc. Those having a determined base sequence can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

In order to enhance the expression level of the ptsS gene, for example, the above DNA may be incorporated into a plasmid capable of functioning in the host microorganism so as to be expressed and introduced into the host microorganism.
The plasmid vector into which the ptsS gene can be incorporated is not particularly limited as long as it contains at least a gene that controls the replication growth function in the host bacterium. Specific examples of plasmids that can be used to introduce genes into coryneform bacteria include plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262. Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in the publication; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; PHM1519 described in JP-A-77895; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197 It can be mentioned pCG4 and pCG11 like described in JP 57-183799 JP.
Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX are preferably used.

In addition, DNA cleavage, ligation, and other methods such as chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc. adopt ordinary methods well known to those skilled in the art. can do. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989), etc.

A recombinant vector obtained by inserting the ptsS gene into an appropriate site of a plasmid vector that can replicate in the coryneform bacterium as described above, such as a coryneform bacterium such as Brevibacterium flavum MJ-233 strain By transforming (FERM BP-1497), coryneform bacteria with increased expression of the ptsS gene can be obtained.
Transformation is performed by, for example, the electric pulse method (Res. Microbiol., Vol. 144, p.181-185, 1993).
Etc.
Further, the expression level of the ptsS gene can be enhanced by making multiple copies of the ptsS gene on the chromosome by a known homologous recombination method.
Further, the expression level of the ptsS gene can be enhanced by replacing or modifying the promoter of the ptsS gene on the host chromosome. Examples of the promoter replacement method include a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73).

The promoter for expressing the ptsS gene or the promoter used for replacing the promoter of the ptsS gene on the chromosome in the introduction by the above-described recombinant plasmid or homologous recombination on the chromosome can function in the host bacteria. The promoter is not particularly limited, but a promoter whose transcriptional activity is not suppressed under anaerobic conditions is preferable, and examples thereof include a tac promoter used in E. coli and a trc promoter.
Thus, the expression level of the ptsS gene can be controlled by appropriately selecting a promoter.
As mentioned above, although the example using coryneform bacteria was described, also when using other bacteria, the expression enhancement of a ptsS gene can be achieved by the same method.

The bacterium of the present invention may be a bacterium modified so that lactate dehydrogenase (also referred to as LDH) activity is reduced in addition to the enhanced expression of the ptsS gene. Here, “LDH activity is reduced” means that LDH activity is reduced as compared to an unmodified strain. The LDH activity is preferably reduced to 10% or less per unit cell weight as compared to the unmodified strain. LDH activity may be completely absent. The decrease in LDH activity can be confirmed by measuring LDH activity by a known method (L. Kanarek and RL Hill, J. Biol. Chem. 239, 4202 (1964)). As a specific method for producing a strain having a reduced LDH activity of a coryneform bacterium, a method by homologous recombination to a chromosome described in JP-A-11-206385 or a method using a sacB gene (Schafer, A. et al. Gene 145 (1994)
69-73). A coryneform bacterium with reduced LDH activity and enhanced expression of the ptsS gene can be obtained, for example, by producing a bacterium with a disrupted LDH gene and transforming the bacterium with a recombinant vector containing the ptsS gene. it can. However, either the modification operation for reducing LDH activity or the modification operation for enhancing expression of the ptsS gene may be performed first.

The bacterium used in the present invention may be a bacterium modified so that the activity of pyruvate carboxylase (hereinafter also referred to as PC) is enhanced in addition to the enhanced expression of the ptsS gene. “The PC activity is enhanced” means that the PC activity is preferably increased by 1.5 times or more, more preferably by 3 times or more per unit cell weight relative to an unmodified strain such as a wild strain or a parent strain. Say. The enhanced PC activity can be confirmed by measuring the PC activity by a known method (Magasanik's method [J. Bacteriol., 158, 55-62, (1984)]). Such a bacterium can be obtained, for example, by introducing the pc gene into a coryneform bacterium having enhanced expression of the ptsS gene. Any modification operation for enhancing PC activity and ptsS gene expression may be performed first.

The introduction of the pc gene can be performed in the same manner as the introduction of the ptsS gene. Specifically, for example, it can be carried out by highly expressing the pc gene in coryneform bacteria in the same manner as described in JP-A-11-196888. As a specific pc gene, for example, a pc gene derived from Corynebacterium glutamicum (Peters-Wendisch, PG et al. Microbiology, vol. 144 (1998) p915-927) can be used. Further, the pc gene is DNA that hybridizes with the pc gene derived from Corynebacterium glutamicum under stringent conditions, or the base sequence of the gene is 80% or more, preferably 90% or more, more preferably 95% or more. Particularly preferably, DNA having a homology of 99% or more and encoding a protein having PC activity can also be suitably used.
Furthermore, pc genes derived from coryneform bacteria other than Corynebacterium glutamicum, or from other microorganisms or animals and plants can also be used. In particular, the microorganism or animal or plant-derived pc gene shown below has a known sequence (shown below), and by amplifying the ORF portion by hybridization or PCR as described above, Can be acquired.
Human [Biochem.Biophys.Res.Comm., 202, 1009-1014, (1994)]
Mouse [Proc.Natl.Acad.Sci.USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast; Saccharomyces cerevisiae
[Mol.Gen.Genet., 229, 307-315, (1991)]
Schizosaccharomyces pombe
[DDBJ Accession No .; D78170]
Bacillus stearothermophilus
[GENE, 191, 47-50, (1997)]
Rhizobium etli
[J. Bacteriol., 178, 5960-5970, (1996)]

Furthermore, in addition to enhancing the expression of the ptsS gene, the bacterium of the present invention comprises acetate kinase (hereinafter also referred to as ACK), phosphotransacetylase (hereinafter also referred to as PTA), pyruvate oxidase (hereinafter also referred to as POXB). And a bacterium modified so as to reduce the activity of one or more enzymes selected from the group consisting of acetyl-CoA hydrolase (hereinafter also referred to as ACH).

Either PTA or ACK may decrease the activity, but it is more preferable to decrease both activities in order to efficiently reduce the byproduct of acetic acid.
“PTA activity” refers to the activity of catalyzing the reaction of transferring phosphoric acid to acetyl-CoA to produce acetyl phosphate. “Modified to reduce PTA activity” means that the PTA activity is lower than that of an unmodified strain such as a wild strain. The PTA activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less per unit cell weight, compared to the unmodified strain. Moreover, PTA activity may be completely lost. The decrease in PTA activity is based on the method of Klotzsch et al. (Klotzsch, HR, Meth Enzymol. 12, 381-386 (1969)).
Can be confirmed by measuring the PTA activity.

“ACK activity” refers to the activity of catalyzing the reaction of producing acetic acid from acetyl phosphate and ADP. “Modified to reduce ACK activity” means that ACK activity is lower than that of an unmodified strain, for example, a wild strain. The ACK activity is preferably reduced to 30% or less per microbial cell, and more preferably 10% or less per microbial cell, compared to the unmodified strain. Further, the ACK activity may be completely lost. The decrease in ACK activity can be confirmed by measuring the ACK activity by the method of Ramponi et al. (Ramponi G., Meth. Enzymol. 42, 409-426 (1975)).

In Corynebacterium glutamicum (including those classified as Brevibacterium flavum), both enzymes are pta as described in Microbiology. 1999 Feb; 145 (Pt 2): 503-13. Since it is encoded by the ack operon (GenBank Accession No. X89084), when the pta gene is disrupted, the activities of both PTA and ACK enzymes can be reduced.

The decrease in PTA and ACK activity can be achieved by disrupting these genes according to known methods, for example, using homologous recombination or using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). Can be done. Specifically, it can be performed according to the method disclosed in JP-A-2006-000091 and the examples described later. As the pta gene and the ack gene, in addition to the gene having the base sequence of the above GenBank Accession No. X89084, a gene having a degree of homology that causes homologous recombination with the pta gene and the ack gene on the host chromosome can also be used. . Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions.

“POXB activity” refers to the activity of catalyzing the reaction of producing acetic acid from pyruvic acid and water. “Modified to reduce POXB activity” means that POXB activity is lower than that of an unmodified strain such as a wild strain. The POXB activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. “Decrease” includes the case where the activity is completely lost. The decrease in POXB activity can be confirmed by measuring POXB activity by the method of Chang et al. (Chang Y. and Cronan J. E. JR, J. Bacteriol. 151, 1279-1289 (1982)).

The decrease in POXB activity can be achieved by disrupting the poxB gene according to a known method such as a method using homologous recombination or a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). It can be carried out. Specifically, it can be performed according to the methods disclosed in WO2005 / 113745 and the examples described later. Examples of the poxB gene include GenBank Accession No. A gene having the base sequence of Cgl2610 (the complementary strand of 2776766-2778505 of GenBank Accession No. BA000036) is mentioned, but it has homology to the extent that homologous recombination occurs with the poxB gene on the chromosomal DNA of the host bacterium. Therefore, a homologous gene of the sequence can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions.

“ACH activity” refers to the activity of catalyzing the reaction of producing acetic acid from acetyl CoA. “Modified to reduce ACH activity” means that ACH activity is lower than that of a non-modified strain such as a wild-type strain. The ACH activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. The “decrease” includes the case where the activity is completely lost. ACH activity can be measured by the method of Gergely, J., et al. (Gergely, J., Hele, P. & Ramkrishnan, CV (1952) J. Biol. Chem. 198 p323-334).

The decrease in ACH activity can be achieved by disrupting the ach gene according to a known method, for example, a method using homologous recombination or a method using the sacB gene (Schafer, A. et al. Gene 145 (1994) 69-73). It can be carried out. Specifically, it can be carried out according to the methods disclosed in WO2005 / 113744 and examples described later. Examples of the ach gene include GenBank Accession No. The gene has the base sequence of Cgl2569 (the 2729376..2730917 complementary strand of GenBank Accession No. BA000036). Therefore, a homologous gene of the sequence can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions.

In addition, the bacterium used in the present invention may be a bacterium obtained by combining two or more of the above modifications in addition to the modification that enhances the expression level of the ptsS gene. When making a plurality of modifications, the order does not matter.

When using the bacterium in the production reaction of a non-amino organic acid such as succinic acid, a slant culture in a solid medium such as an agar medium may be used for the direct reaction. However, the bacterium is previously cultured in a liquid medium (seed It is preferable to use those cultured. The bacteria thus seed-cultured can be produced by reacting with sucrose while growing on a medium containing sucrose. Moreover, it can manufacture also by making the microbial cell obtained by making it grow react with sucrose in the reaction liquid containing sucrose. In order to use an aerobic coryneform bacterium in the method of the present invention, it is preferable to first use the cells after culturing them under normal aerobic conditions. As a medium used for culture, a medium usually used for culture of microorganisms can be used. For example, a general medium in which a natural nutrient source such as meat extract, yeast extract or peptone is added to a composition composed of inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used. The cultured cells are collected by centrifugation, membrane separation, etc. and used for the reaction.

In the present invention, a treated product of bacterial cells can also be used. Examples of treated cells include, for example, immobilized cells obtained by immobilizing cells with acrylamide, carrageenan, etc., crushed materials obtained by crushing cells, centrifugation supernatants thereof, or partial supernatants thereof by ammonium sulfate treatment, etc. Examples include purified fractions.

The concentration of sucrose used is not particularly limited, but it is advantageous to make it as high as possible as long as it does not inhibit the production of non-amino organic acid, usually 5 to 30% (W / V), preferably 10 to 20%. The reaction is carried out within the range of (W / V). Further, additional sucrose may be added as the sucrose decreases with the progress of the reaction.

The reaction solution containing the sucrose is not particularly limited, and may be a medium for culturing bacteria or a buffer solution such as a phosphate buffer. The reaction solution is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by the microorganism to produce a non-amino organic acid. Specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein Various organic and inorganic nitrogen compounds such as degradation products, peptone, yeast extract, meat extract, corn steep liquor and the like can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, factors that promote growth such as vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, and amino acids are added as necessary. In order to suppress foaming during the reaction, it is desirable to add an appropriate amount of a commercially available antifoaming agent to the culture solution.

The pH of the reaction solution can be adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide or the like. Since the pH in this reaction is usually pH 5 to 10, preferably 6 to 9.5, the pH of the reaction solution is within the above range depending on the alkaline substance, carbonate, urea, etc. as necessary even during the reaction. Adjust to.

As the reaction solution used in the present invention, water, a buffer solution, a medium, or the like is used, and a medium is most preferable. The medium can contain, for example, sucrose and carbonate ions, bicarbonate ions, or carbon dioxide gas, and can be reacted under anaerobic conditions. Carbonate ions or bicarbonate ions are supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, which can also be used as a neutralizing agent. It is also possible to supply from the salt or carbon dioxide gas. Specific examples of the carbonate or bicarbonate salt include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, and potassium bicarbonate. Carbonate ions and bicarbonate ions are added at a concentration of 1 to 500 mM, preferably 2 to 300 mM, more preferably 3 to 200 mM. When carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide gas is contained per liter of the solution.

The optimal growth temperature for bacteria used in this reaction is usually 25 ° C to 35 ° C. The temperature during the non-amino organic acid production reaction is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. Although the quantity of the microbial cell used for reaction is not prescribed | regulated, 1-700 g / L, Preferably it is 10-500 g / L, More preferably, 20-400 g / L is used. The reaction time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.

When culturing bacteria (cell growth), it is necessary to supply oxygen by aeration and agitation. On the other hand, the production reaction of the non-amino organic acid may be performed by aeration and stirring, but may be performed in an anaerobic atmosphere without aeration and without supplying oxygen. In this case, an anaerobic atmosphere may be obtained by a method such as sealing the container and reacting without aeration, supplying an inert gas such as nitrogen gas, reacting, or venting an inert gas containing carbon dioxide gas. it can.

The non-amino organic acid accumulated in the reaction solution (culture solution) can be recovered (separated and purified) from the reaction solution according to a conventional method. Specifically, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin or the like, and separating and purifying the non-amino organic acid from the solution by crystallization or column chromatography be able to.

Further, in the present invention, after producing a non-amino organic acid such as succinic acid by the above-described method of the present invention, a non-amino organic acid-containing polymer is produced by performing a polymerization reaction using the obtained non-amino organic acid as a raw material. can do. In recent years, with the increase in the number of environmentally friendly industrial products, attention has been focused on polymers using plant-derived raw materials. In particular, succinic acid produced in the present invention is processed into polymers such as polyester and polyamide. Can be used. Specific examples of succinic acid-containing polymers include succinic acid polyesters obtained by polymerizing diols such as butanediol and ethylene glycol and succinic acid, succinic acid polyamides obtained by polymerizing diamines such as hexamethylenediamine and succinic acid, and the like. Is mentioned. The non-amino organic acid obtained by the production method of the present invention or the composition containing the non-amino organic acid can be used for food additives, pharmaceuticals, cosmetics and the like.

When the non-amino organic acid is succinic acid, a succinic acid derivative can be produced using the obtained succinic acid after producing succinic acid by the method of the present invention. Here, as the succinic acid derivative, succinic acid salt, succinic anhydride, maleic anhydride, succinic acid ester, succinimide, 1,4-diaminobutane, succinic acid nitrile, 2-pyrrolidone, N-methylpyrrolidone (NMP) N-ethylpyrrolidone (NEP), 1,4-butanediol (1,4-BG), tetrahydrofuran (THF), γ-butyrolactone (GBL), polytetramethine ether glycol (PTMG) and the like. For example, 1,4-butanediol can be produced by hydrogenating succinic acid. 1,4-Butanediol is used as a raw material for polybutylene succinate resin, polybutylene terephthalate resin, tetrahydrofuran (solvent), γ-butyrolactan, which is a precursor of N-methylpyrrolidone and N-vinylpyrrolidone.

[Example]
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

<Preparation of ptsS enhanced strain>
(A) Extraction of genomic DNA of Brevibacterium flavum MJ233 strain A medium [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 0.5 g O, 6 mg FeSO ₄ .7H ₂ O, 6 mg MnSO ₄ · 4-5H ₂ O, 200 μg biotin, 100 μg thiamine, 1 g yeast extract, 1 g casamino acid, 20 g glucose, 1 L distilled water] in 10 mL, Brevi The bacterial flavum strain MJ233 was cultured until the late logarithmic growth phase, and the cells were collected by centrifugation (10000 g, 5 minutes). The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. To this lysate, add an equal amount of phenol / chloroform solution, shake gently at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain a supernatant. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was 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 left overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Construction of plasmid for ptsS gene promoter replacement Obtaining the DNA fragment of the N-terminal region of the ptsS gene derived from Brevibacterium flavum MJ233 strain has been reported using the DNA prepared in (A) above as a template and the entire genome sequence. It was carried out by PCR using synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed on the basis of the gene sequence (GenBank Database Accession No. AP005276) of Corynebacterium glutamicum ATCC13032 strain. Composition of reaction solution: Template DNA 1 μL, Ex-Taq DNA polymerase (manufactured by Takara Bio Inc.) 0.2 μL, 1-fold concentration buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 4 minutes. Confirmation of the amplification product is carried out by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining.
An approximately 0.6 kb fragment was detected and used as the ptsS gene N-terminal fragment.

  On the other hand, the TZ4 promoter fragment derived from Brevibacterium flavum MJ233 strain is constitutively highly expressed using plasmid pMJPC17.2 (Japanese Patent Application Laid-Open No. 2005-95169) as a template and using the synthetic DNAs described in SEQ ID NO: 3 and SEQ ID NO: 4. Prepared by PCR. Composition of reaction solution: Template DNA 1 μL, Ex-Taq polymerase (manufactured by Takara Bio Inc.) 0.2 μL, 1 × concentration attached buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 3 minutes. Confirmation of the amplification product is performed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMCBioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.2 kb is detected, which is detected as TZ4. A promoter fragment was used.

A DNA fragment in which the TZ4 promoter and the ptsS gene N-terminal region were linked was obtained by crossover PCR using the synthetic DNAs of SEQ ID NO: 5 and SEQ ID NO: 6 using both of the fragments prepared above as templates. Reaction solution composition: template DNA 1 μL, Ex-Taq each
DNA polymerase (manufactured by Takara Bio Inc.) 0.2 μL, 1 × concentration attached buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 4 minutes. Confirmation of the amplification product was performed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.8 kb was detected. The obtained DNA fragment in which the TZ4 promoter and the ptsS gene N-terminal region were linked was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN) and then digested with restriction enzymes EcoRI and KpnI. This DNA fragment was mixed with Escherichia coli vector pHSG299 (manufactured by Takara Bio Inc.) treated with the same restriction enzyme, and ligation kit ver. 2 (manufactured by Takara Bio Inc.). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with restriction enzymes EcoRI and KpnI, and an inserted fragment of about 0.8 kb was recognized. This was named pHSGptsS.

(C) Production of ptsS-enhanced strain by promoter replacement Brevibacterium flavum MJ233 / PC-4 / ΔLDH (Strain in which expression of pyruvate carboxylase gene is enhanced and lactate dehydrogenase gene is disrupted: JP-A-2005-95169) Plasmid DNA used for transformation was re-prepared from E. coli JM110 strain transformed with the above pHSGptsS plasmid DNA by the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970). Transformation of the Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol. Vol. 144, p. 181-185, 1993), and the obtained transformant was treated with 25 μg / kanamycin. LBG agar medium containing mL [trypton 10 g, yeast extract 5 g, NaCl
5 g, glucose 20 g, and agar 15 g were dissolved in 1 L of distilled water]. Since the strain grown on this medium is a plasmid in which pHSGptsS cannot replicate in the Brevibacterium flavum MJ233 strain, the ptsS gene of the plasmid and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination, a kanamycin resistance gene derived from the plasmid should be inserted on the genome. Confirmation that the promoter of the ptsS gene on the genome has been replaced by the TZ4 promoter is to directly subject the cells obtained by liquid culture in LBG medium to a PCR reaction and detect the TZ4 promoter and the ptsS gene region. Can be confirmed. When these regions are analyzed using primers (SEQ ID NO: 7 and SEQ ID NO: 8) for PCR amplification, a 608 bp DNA fragment should be observed in the promoter-substituted form. As a result of analyzing the kanamycin resistant strain by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / PtsS / PC-4 / ΔLDH.

<Evaluation of ptsS enhanced strain>
Seed culture medium (urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate: 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate, heptahydrate Product: 20 mg, manganese sulfate / hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, distilled water: made up to 1000 mL) 100 mL is put into a 500 mL Erlenmeyer flask And sterilized by heating at 120 ° C. for 20 minutes. This was cooled to room temperature, 4 mL of 50% sucrose aqueous solution sterilized by a 0.22 μm membrane filter (MILLIPORE), and 50 μL of 5% kanamycin aqueous solution filtered aseptically were added, and the Brevibacterium prepared in Example 1 (C) was added. Inoculation with Um flavum MJ233 / PtsS / PC-4 / ΔLDH strain or Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain (JP 2005-95169) as a control strain and seed culture at 30 ° C. for 16 hours did. However, MJ233 / PC-4 / ΔLDH strain was cultured without kanamycin.

The obtained whole culture solution was collected by centrifugation at 4000 × g for 7 minutes, and a cell suspension medium (magnesium sulfate 7-hydrate: 1 g, ferrous sulfate 7-hydrate: 40 mg, sulfuric acid Manganese hydrate: 40 mg, D-biotin: 400 μg, thiamine hydrochloride: 400 μg, monoammonium phosphate: 0.8 g, diammonium phosphate: 0.8 g, potassium chloride: 0.3 g, ammonium sulfate 66 g, distilled water: In order to make the absorbance of OD ₆₆₀ 20 in 1000 mL). Add 0.5 mL of the substrate solution (sucrose: 40 g, ammonium hydrogen carbonate: 63 g, and distilled water: 1000 mL) to 0.5 mL of the above cell suspension in a 4 mL reactor, 20% carbon dioxide, 80% nitrogen The reaction was carried out at 39 ° C. for 6 hours under atmosphere.

  After the reaction, the mixture was centrifuged under the conditions described above, and the organic acid concentration in the supernatant was analyzed. As a result, the yield of succinic acid per sucrose consumed by the control strain Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain (weight) ) Was 67%, whereas the Brevibacterium flavum MJ233 / PtsS / PC-4 / ΔLDH strain was 83%, and the enhancement of ptsS was effective in improving the yield of succinic acid to sugar. It has been shown.

Claims (7)

Modified to produce non-amino organic acid selected from succinic acid and acetic acid , enhance ptsS gene expression compared to non-modified strain, and reduce lactate dehydrogenase activity compared to non-modified strain Bacteria. The ptsS gene has a sequence in which 1 to 5 amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10, and encodes a protein having PtsS activity. The bacterium according to claim 1. The bacterium according to claim 1 or 2 , further modified so that pyruvate carboxylase activity is enhanced as compared with an unmodified strain. The bacterium according to any one of claims 1 to 3 , which is a coryneform bacterium. The succinic acid and succinic acid in a reaction liquid by making the bacteria as described in any one of Claims 1-4 , or its processed material act on sucrose in the reaction liquid containing a carbonate ion, bicarbonate ion, or a carbon dioxide gas. A method for producing a non-amino organic acid selected from succinic acid and acetic acid, comprising producing and accumulating a non-amino organic acid selected from acetic acid and collecting the non-amino organic acid. The method for producing a succinic acid-containing polymer, comprising a step of producing succinic acid by the method according to claim 5 and a step of polymerizing the succinic acid obtained in the step, wherein the non-amino organic acid is succinic acid . A non-amino organic acid is succinic acid, comprising a step of producing succinic acid by the method according to claim 5 and a step of synthesizing a succinic acid derivative using succinic acid obtained in the step as a raw material. Production method.