JP5796282B2 - Chemical production method using coryneform bacteria - Google Patents

Description

  The present invention is a method for producing a chemical product by culturing coryneform bacteria having the ability to produce a chemical product, filtering the obtained fermentation broth with a separation membrane, and recovering the product from the filtrate. The present invention relates to a method for producing a chemical product comprising adding a cell wall synthesis inhibitor to a medium.

  Coryneform bacteria have been conventionally used in the production of chemicals such as L-lysine, L-glutamic acid, L-amino acids such as L-tryptophan, polyamines such as cadaverine, and organic acids such as succinic acid. (See Patent Documents 1 to 5.) Fermentation methods, which are chemical production methods using coryneform bacteria, are roughly classified into batch fermentation methods (Batch fermentation methods), fed-batch fermentation methods (Fed-Batch fermentation methods), and continuous fermentation methods.

  The batch fermentation method and the fed-batch fermentation method have the advantage that they are simple in terms of equipment, complete the culture in a short time, and are less damaged by contamination with various bacteria. However, in order to recover chemicals from the fermentation broth after completion of fermentation in batch fermentation and fed-batch fermentation, it is necessary to remove bacterial cells in the fermentation broth for transfer to the subsequent purification process. As a solid-liquid separation method for removing bacterial cells from the fermentation broth, filtration or separation by a separation membrane is required. In addition, solid-liquid separation of the fermentation broth by centrifugation requires time, and the longer the time for solid-liquid separation, the lower the operation rate of the fermentation process and the higher the possibility of contamination with foreign bacteria. Since the quality of products can change, recently, filtration separation using a separation membrane that can shorten the time of solid-liquid separation that leads to efficient fermentation process and prevention of contamination with bacteria has been studied.

  On the other hand, continuous fermentation, particularly continuous fermentation using a separation membrane, can be stably and efficiently cultured and produced over a long period of time by continuous filtration using a separation membrane of the fermentation broth containing the target chemical product, There is an advantage that high productivity can be maintained by continuous fermentation (Patent Document 6).

JP 2007-514439 A JP 2008-212138 A JP 2008-200046 A Japanese Patent Application Laid-Open No. 2004-222569 JP 2008-0667629 A JP 2008-104453 A

  In order to shorten the solid-liquid separation time of the fermentation liquid and improve the productivity of the chemical product in the method for producing a chemical product by fermentation using a separation membrane, the present inventor filtered the fermentation solution by improving the clogging of the membrane. It was found that there is a need to improve performance. Accordingly, the present invention provides a method for producing a chemical product by fermentation of coryneform bacteria by improving filterability when filtering the fermentation broth using coryneform bacteria with a separation membrane by a simple method. It is an object to shorten time for liquid separation and improve productivity.

As a result of intensive studies by the present inventors in order to solve the above problems, it has been found that the problems of the present invention can be solved by adding a cell wall synthesis inhibitor to the fermentation medium, and the present invention has been completed. That is, this invention is comprised by the following (1)-( 7 ).

(1) Coryneform bacteria having the ability to produce chemicals are cultured in a fermentation medium containing a mycolic acid synthesis inhibitor or a drug that suppresses mycolic acid adhesion to the cell wall, and the resulting fermentation broth is averaged as a separation membrane A method for producing a chemical product, comprising filtering a porous membrane having pores having a pore diameter of 0.01 μm or more and less than 1 μm and recovering a product from the filtrate.

( 2 ) The method for producing a chemical according to (1 ), wherein the mycolic acid synthesis inhibitor or the drug that suppresses mycolic acid adhesion to a cell wall is isonicotinic acid hydrazide (INH) or ethambutol.

(3) the content of the mycolic acids adhering agent that inhibits to mycolic acid synthesis inhibitors or cell wall in the fermentation medium is characterized in that it is a 0.1 to 20 g / L, (1) or (2) A method for producing the chemical product according to 1.

(4) culturing the coryneform bacterium, the unfiltered liquid of the filtration held or reflux in the fermentation broth, and, characterized in that the fermentation medium is continuously continuous fermentation culture added , (1)-( 3 ) The manufacturing method of the chemical in any one of ( 3 ).

( 5 ) The method for producing a chemical product according to any one of (1) to ( 4 ), wherein the coryneform bacterium is a genus Corynebacterium or Brevibacterium.

( 6 ) The method for producing a chemical product according to any one of (1) to ( 5 ), wherein the chemical product is an amino acid or a polyamine.

( 7 ) The method for producing a chemical product according to any one of (1) to ( 6 ), wherein the chemical product is L-lysine or cadaverine.

  According to the present invention, by adding a cell wall synthesis inhibitor to the fermentation medium, the filterability of the fermentation broth can be greatly improved during the fermentation of coryneform bacteria. The improvement in the filterability of the fermentation broth can reduce the time of the fermentation broth or improve the filtration rate because the separation membrane is less likely to be clogged during the solid-liquid separation of cells from the fermentation broth. In addition, during continuous production of chemicals using coryneform bacteria, the separation membrane is less likely to be clogged, thereby improving the productivity of chemicals over long periods of operation or reducing the area of the separation membrane used for continuous fermentation. The fermentation apparatus can be made compact.

FIG. 1 is a graph comparing the filtration time and transmembrane pressure difference between Example 1 and Comparative Example 1. FIG. 2 is a graph comparing the filtration time and transmembrane pressure in Example 2 and Comparative Example 2. FIG. 3 is a graph comparing the filtration time and the transmembrane pressure in Example 3 and Comparative Example 3. FIG. 4 is a graph comparing the filtration time and the transmembrane pressure in Example 4 and Comparative Example 4. FIG. 5 is a graph comparing the culture time and transmembrane pressure in Example 5 and Comparative Example 5. FIG. 6 is a graph comparing the culture time and transmembrane pressure in Example 6 and Comparative Example 6. FIG. 7 is a graph comparing the culture time and transmembrane pressure difference between Example 7 and Comparative Example 7. FIG. 8 is a graph comparing the culture time and transmembrane pressure in Example 8 and Comparative Example 8.

  The present invention is characterized by culturing coryneform bacteria having an ability to produce a chemical product to obtain a fermentation broth containing the chemical product.

  Coryneform bacteria are bacteria belonging to Gram-positive isolated as amino acid-producing bacteria in the 1950s. The coryneform bacterium used in the present invention is not particularly limited as long as it grows under normal aerobic conditions and produces the chemical product targeted by the present invention. As a specific example, Agrococcus ( Agrococcus genus, Agromyces genus, Arthrobacter genus, Aureobacterium genus, Brevibacterium genus, Cellulomonas genus, Clavibacter bacterium Corynebacterium, Microbacterium, Rathaybacter, Terrabacter ) Genus Tsurisera (Turicella) include bacteria belonging to the genus, preferably a bacterium belonging to the genus Corynebacterium or Brevibacterium, or more preferably a bacterium belonging to the genus Corynebacterium. Specific examples of bacteria belonging to the genus Corynebacterium include Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 13870, Corynebacterium carnae ATCC 15991, Corynebacterium acetoglutamicum. ATCC 15806.

  In the present invention, coryneform bacteria bred by mutant screening and gene recombination, which are methods known to those skilled in the art, are preferably used so that a desired chemical product can be efficiently produced. Examples of chemicals that can be preferably produced by the coryneform bacterium so bred include amino acids or organic acids, nucleic acids, polyamines, vitamins, proteins such as various enzymes, peptides, sugars, sugar alcohols, alcohols, lipids, etc. Are preferably amino acids or polyamines.

  Specific examples of amino acids that can be produced by coryneform bacteria include tryptophan, lysine, methionine, phenylalanine, threonine, valine, isoleucine, leucine, histidine, alanine, arginine, asparagine, serine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, Proline and tyrosine, which may be L-amino acid, D-amino acid, or DL-amino acid.

  Amino acids are produced by fermentation of coryneform bacteria. These coryneform bacteria capable of producing amino acids are coryneform bacteria isolated by screening from the natural world, or mutation operations such as disrupting specific genes or enhancing or decreasing expression [for example, N-methyl-N′- Methods using nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), or mutagenesis by UV irradiation or in vitro mutagenesis ("Molecular and General Genetics", 1978, vol. 145, p. 101)], or coryneform bacteria by inducing mutation in the gene or enhancing or introducing amino acid synthesis ability by gene recombination.

  As an example of enhancing or introducing amino acid synthesis ability of coryneform bacteria by mutation or genetic recombination, a mutation method for introducing or deleting one or more bases in a gene encoding phosphoenolpyruvate carboxykinase (PEPCK), A step of growing Corynebacterium by introduction of a nonsense mutation or a method for reducing the activity of the polypeptide, the step of concentrating L-amino acids in a medium or cells, and the step of separating L-amino acids. A method for producing an amino acid (US Pat. No. 6,872,553), and a method for improving L-amino acid production ability include deletion of an L-amino acid into a cell or a decrease in activity of the system or L-lysine, L- L-amino acid excretion system such as arginine excretion gene (LysE) And the like of (European Patent Application Publication No. 1038970, No. WO01 / 005959, J Mol Microbiol Biotechnol 1999 Nov; 1 (2): 327-36, No. WO97 / 23597).

  Examples of L-lysine production include L-lysine production by increasing pyruvate carboxylase (PC) activity, lacking malate quinone oxidoreductase, etc. WO2003 / 0044943). Also, production using mutants having resistance to S- (2-aminoethyl) -cysteine (AEC), etc., strains that require one or more amino acids such as L-homoserine, or AEC and one or more amino acids are required for the production. (Japanese Patent Publication No. 56-1914, Japanese Patent Publication No. 56-1915, Japanese Patent Publication No. 57-14157, Japanese Patent Publication No. 57-14158, Japanese Patent Publication No. 57-30474, Japanese Patent Publication No. 58-10075). No. 59, JP-B 59-4993, JP-B 61-35840, JP-B 62-24074, JP-B 62-36673, JP-B 5-11958, JP-B 7-112437, JP-B 7-11438 No. 48-28078, No. 56-6499, US Pat. Nos. 3,708,395 and 3,825,472). In addition, a strain having a mutation introduced so as to be resistant to DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartic acid-analog, sulfa drug, quinoid, N-lauroylleucine, oxaloacetate decarboxylase Alternatively, mutant strains exhibiting resistance to respiratory enzyme inhibitors can be mentioned (Japanese Patent Laid-Open Nos. 50-53588, 50-31093, 52-102498, 53-9394, and JP-A 53-9394). JP 53-86089, JP 55-9783, JP 55-9759, JP 56-32995, JP 56-39778, JP 53-43591, JP 53-1833 issue). In addition, mutants showing the requirement for one or more combinations of vitamins or organic acids such as inositol or acetic acid, fluoropyruvic acid, mutants showing temperature sensitivity to temperatures of 34 ° C. or higher, ethylene glycol resistant strains, etc. (Japanese Patent Laid-Open Nos. 55-9784, 56-8692, 55-9783, 53-86090, US Pat. No. 4,411,997). Moreover, the microorganisms provided with the ability to produce L-lysine can also be obtained by enhancing the activity of the enzyme involved in the L-lysine biosynthetic pathway. These increases in enzyme activity can be achieved by increasing the copy number of the gene encoding the enzyme in the cell and changing the expression regulatory sequence. Furthermore, the activity of an enzyme that catalyzes a reaction for producing a substance other than L-lysine and the enzyme activity that functions negatively for L-lysine production may be reduced or lost (WO95 / 23864, WO96 / 17930, WO 2005/010175).

  In L-glutamine production, coryneform bacteria producing L-glutamine by culture containing biotin in the medium, L-glutamine-producing coryneform bacteria having purine analog resistance and / or methionine sulfoxide resistance, and L-glutamine production A coryneform bacterium having an ability to increase glutamine synthetase activity, or a coryneform bacterium modified so that intracellular glutaminase activity is decreased and intracellular glutamine synthetase activity is enhanced (particularly). JP-A-62-186796, JP-A-61-202694, JP-A-2002-300887, JP-A-2004-187684). Furthermore, as a method for increasing the L-glutamic acid concentration, a method for deleting a gene encoding α-ketoglutarate dehydrogenase (WO95 / 34672) or a method for enhancing a gene encoding glutamine synthetase (Japanese Patent Laid-Open No. 2002-300887). ), Glutamate dehydrogenase gene (gdh), isocitrate dehydrogenase gene (icdA), aconitic acid hydratase gene (acnA, acnB), and citrate synthase gene (gltA) are disclosed (Japanese Patent Laid-Open No. Sho 63-63). 214189). In addition, L-glutamic acid production is induced by biotin restriction, surfactant addition, penicillin addition, etc., or glutamate production is performed by surfactant temperature sensitive or penicillin sensitive strains (European patent application) (Publication No. 100266, JP-A-4-089944).

In L-alanine fermentation, strains with increased alanine production by homologous recombination of L-alanine dehydrogenase gene, strains deficient in H ⁺ -ATPase activity, and aspartate β-decarboxylase gene were amplified. (Japanese Patent Laid-Open No. 6-277082, Appl Microbiol Biotechnol. 2001 Nov; 57 (4): 534-40, Japanese Patent Laid-Open No. 7-163383).

  In the production of L-tryptophan, L-phenylalanine and L-tyrosine are required, and 4-methyl-tryptophan, 6-fluoro-tryptophan, 4-amino-phenylalanine, 4-fluoro-phenylalanine, tyrosine-hydroxamate, phenylalanine -Tryptophan production by Corynebacterium glutamicum ATCC 21851 having resistance to hydroxamate is mentioned (Japanese Patent Publication No. 7-59199). Examples thereof include coryneform bacteria in which one or more of the activities of tryptophan synthase activity, anthranilate synthase activity and phosphoglycerate dehydrogenase activity are enhanced (WO94 / 08031 or US Pat. No. 4,371,614). Furthermore, by introducing a recombinant DNA containing a tryptophan operon, L can be produced by introducing a mutation that imparts the ability to produce L-tryptophan, or deletes the gene encoding the repressor of the tryptophan operon, or causes a decrease in the activity of the repressor. -Tryptophan production can be imparted (JP 57-71397, JP 62-244382, US Pat. No. 4,371,614, WO 2005/056776). In addition, malate synthase, isocitrate lyase, isocitrate dehydrogenase kinase / phosphatase operon are constitutively expressed or expression of the operon is enhanced. A strain resistant to sulfaguanidine, a strain introduced with a tryptophan operon, or a strain introduced with a gene encoding shikimate kinase can be used (Japanese Patent Publication No. 3-14436, Japanese Patent Laid-Open No. 63-240794). JP, 61-78378, A).

  In L-arginine production, it has drug resistance such as sulfa drugs, 2-thiazolealanine, α-amino-β-hydroxyvaleric acid by mutagenesis, L-histidine, L-proline, L-threonine, L-isoleucine, Examples include L-methionine or L-tryptophan auxotrophic strains, strains resistant to ketomalonic acid, fluoromalonic acid or monofluoroacetic acid, and L-arginine production by strains having arginine resistance. No. 44096, JP-A 57-18989, JP-A 62-24075). Further, by introducing a DNA fragment containing genes of acetylornithine deacetylase, N-acetylglutamate-γ-semialdehyde dehydrogenase, N-acetylglutamokinase, and argininosuccinase derived from E. coli into coryneform bacteria, L -Enhancement of arginine biosynthetic enzyme (Japanese Patent Publication No. 5-23750). Furthermore, as a coryneform bacterium that produces L-arginine, a mutation is introduced so that one or more of strains, amino acids, or precursors thereof are introduced so as to have resistance to an analog such as histidine or tryptophan. So as to be resistant to succinic acid-requiring or nucleobase analogs, strains that have been mutated to have resistance to azapyrimidine derivatives, etc., arginine hydroxamate, and / or 2-thiouracil Breeding to strains with mutations, lacking arginine resolution, resistant to arginine antagonists and canavanine, requiring lysine, and having one or more of the above resistance and requirements And coryneform bacteria (Japanese Patent Laid-Open Nos. 52-114092, 52-99289, HirakiAkira 58-9692, JP No. 49-126819, JP-B-51-6754, JP-A-52-8729, JP-A-53-143288, JP-A-53-3586).

  In the production of L-threonine, a method for producing threonine by recombining the threonine operon (Japanese Patent Publication No. 1-38475) or an improved method after attenuation of the gene encoding serine hydroxymethyltransferase (Japanese Patent Laid-Open No. 2001-190296). ) And the like.

  In L-leucine production, 2-thiazolealanine and β-hydroxyleucine resistance, or valine analog resistance, or valine requirement, or S- (2-aminoethyl) -L-cysteine (AEC) resistance, or phenylalanine, valine And production by coryneform bacteria exhibiting isoleucine requirement (Japanese Patent Laid-Open No. 8-266295, Japanese Patent Laid-Open No. 63-248392, Japanese Patent Publication No. 38-4395, Japanese Patent Publication No. 51-37347, Japanese Patent Publication No. 54-36233). ).

  In L-isoleucine production, a nucleotide sequence encoding the elimination of branched amino acids is amplified, L-isoleucine production ability is imparted by protoplast fusion, homoserine dehydrogenase enhancement of metabolic pathways, production by coryneform bacteria showing drug resistance. (JP 2001-169788, JP 62-74293, JP 62-91193, JP 62-195293, JP 61-15695, JP 61-15696).

  In the production of L-valine, expression enhancement of genes encoding enzymes involved in the L-valine biosynthetic pathway or mutagenesis can be mentioned (WO 00/50624, amino acid fermentation, academic publication center, pages 397-422, 1986). Year).

  As L-phenylalanine production, strains with reduced phosphoenolpyruvate carboxylase or pyruvate kinase activity (JP-A-1-317395), tyrosine-requiring strains (JP-A-63-105688), etc. can be used. .

In L-methionine production, at least five or more strains that produce methionine in the presence of a methylcap sulfide compound or selected from ask ^fbr , hom ^fbr , metX, metY, metB, metH, metE, metF, and zwf And a strain in which the gene modification causes overexpression of at least 5 or more genes (Japanese translations of PCT publication No. 2009-501548, JP-T 2009-501550). issue). Alternatively, a strain that has been recombined so as to express a heterologous metI gene can be used (Japanese translations of PCT publication No. 2009-501547).

  In L-serine production, a strain capable of producing L-serine having resistance to azaserine or β- (2-thienyl) -DL-alanine, a strain in which at least one of phosphoserine phosphatase activity or phosphoserine transaminase activity is enhanced, A strain that is resistant to azaserine or β- (2-thienyl) -DL-alanine and lacks L-serine resolution and produces L-serine, improved microorganism capable of converting glycine to L-serine Improved strains comprising (1) serine dehumanlatase negative and (2) the amino acid analogs serine hydroxamate, glycine hydroxamate and methionine hydroxamate. And a strain resistant to at least one of the above-mentioned compounds (Japanese Patent Application Laid-Open No. 11-266881) JP-A-11-253187, JP-A-10-248588, JP-A-60-120996).

  In L-cysteine production, the expression regulatory sequence of the gene was modified so that the gene encoding serine acetyltransferase was transformed or the expression of the gene encoding serine acetyltransferase in the cells was enhanced. As a result, coryneform bacteria having an increased intracellular serine acetyltransferase activity, preferably coryneform bacteria in which the L-cysteine degradation system is suppressed (Japanese Patent Application Laid-Open No. 2002-233384) can be mentioned.

  Examples of L-proline production include coryneform bacteria that require at least one of hiboxanthin and inositol for growth, or produce L-proline that is resistant to purine analogs or pyrimidine analogs (Japanese Patent Laid-Open No. Sho 59-). No. 120094, JP-A-59-109188).

  L-tyrosine production requires phenylalanine, a mutant strain resistant to 3-aminotyrosine, p-aminophenylalanine, p-fluorophenylalanine, tyrosine hydroxamate, a mutant strain resistant to m-fluorophenylalanine, L -Recombinant coryneform bacteria in which tyrosine requirement is eliminated. (H. Hagino, K. Nakayama; Agric. Biol. Chem. 37, 2001 (1973), Sugimoto, Nakamura, Tsuchida, Shiio; Agric. Biol. Chem. 37, 2327 (1973), Japanese Patent Laid-Open No. 60-1987. ).

  Specific examples of polyamines that can be produced by coryneform bacteria include cadaverine, spermine, spermidine, or putrescine, or a salt thereof, with cadaverine or a salt thereof being preferred. Cadaverine is also called 1,5-pentanediamine. The cadaverine salt is a salt produced when neutralizing basic cadaverine with an acid, and specific examples thereof include cadaverine dihydrochloride, cadaverine sulfate, and cadaverine dicarboxylate. Cadaverine or a salt thereof (hereinafter simply referred to as cadaverine) is a useful compound as a polymer raw material.

  Polyamines are produced by fermentation of coryneform bacteria. The coryneform bacterium capable of producing these polyamines is a coryneform bacterium isolated by screening from the natural world or a mutation operation such as destroying a specific gene or enhancing or decreasing expression [for example, N-methyl-N′-nitro. -Method using N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) or mutagenesis by UV irradiation or in vitro mutagenesis ("Molecular and General Genetics", 1978, vol. 145, p. 101) )] Increases coryneform bacteria by inducing mutation in the gene or enhancing or introducing polyamine synthesis ability by genetic recombination.

  Examples of enhancing or introducing the ability of coryneform bacteria to synthesize polyamines by mutation manipulation or genetic recombination include, for example, production of putrescine in microorganisms having ornithine decarboxylase activity (increased ODC activity), and increased ODC activity. Examples include production of putrescine by the presence of active N-acetylglutamate formation (Japanese Patent Publication Nos. 2008-505652 and 2008-505651), and production of spermidine in spermidine acetyltransferase-deficient cells, production of putrescine by the spermidine synthesis pathway. Etc.

  In the case of cadaverine, coryneform bacteria originally do not have L-lysine decarboxylase activity, and therefore, a strain that produces cadaverine from L-lysine cannot be obtained by screening for mutant strains of coryneform bacteria. Therefore, in order to obtain a coryneform bacterium having an ability to produce cadaverine, it is necessary to impart L-lysine decarboxylase activity to the coryneform bacterium by genetic recombination. The method for imparting L-lysine decarboxylase activity to coryneform bacteria is not particularly limited, and examples thereof include a method disclosed in Japanese Patent Application Laid-Open No. 2004-222569.

  Further, wild-type strains of coryneform bacteria do not have homoserine auxotrophy, but homoserine auxotrophic mutants (hereinafter also referred to as homoserine auxotrophic strains) produce L-lysine as a precursor of cadaverine. Since it has high property, it is preferably used in the present invention. Methods for obtaining homoserine auxotrophic strains include mutagenesis of wild-type strains by mutagenesis, selection using homoserine auxotrophy as an index, and homologous recombination in the chromosomes of wild-type strains. Examples include a method of deleting homoserine dehydrogenase activity by a method of inserting other genes into a homoserine dehydrogenase gene (hereinafter also referred to as a HOM gene), and a method of deleting the HOM gene by homologous recombination is preferred. As disclosed in JP-A-2222569, L-lysine decarboxylase activity and homoserine auxotrophy can be obtained in coryneform bacteria by deleting the HOM gene with the L-lysine decarboxylase gene (hereinafter also referred to as LDC gene). Since it can provide simultaneously, it is more preferable.

  In addition, the wild-type strain of coryneform bacterium can be obtained as a method for obtaining a strain that is feedback-inhibited by L-lysine, which is a precursor of cadaverine, or L-lysine and L-threonine. The method disclosed in 2004-222569 is mentioned. For example, a wild-type strain was subjected to a mutation operation [for example, a method using N-methyl-N′-nitro-N-nitrosoguanidine (NTG); Microbial Experiment Manual, 1986, p. 131, Kodansha Scientific Co., Ltd.]. Subsequently, a method of obtaining from a mutant strain that has been selected using AEC resistance as an index and has become L-lysine-productive can be mentioned. Suitable mutants include L-lysine-producing bacteria derived from Corynebacterium glutamicum wild-type strain ATCC13032 or a wild-type AK gene, and then the NTG method or in vitro mutation method (“Molecular”). and General Genetics ", 1978, Vol. 145, p. 101), a desensitization type AK gene having mutations in the gene, AEC resistance, and desensitization using L-lysine productivity as an index. Can be included. As a more preferable method for imparting AEC resistance to a wild type strain, a method using a genetic engineering technique as disclosed in JP-A No. 2004-222569 is used. There are no particular limitations on the method using genetic engineering techniques, for example, a method using a recombinant DNA replicable in coryneform bacteria, or a method of recombining a target gene in a chromosome by homologous recombination. It is possible to acquire S- (2-aminoethyl) -L-cysteine resistance by having a mutant aspartokinase gene in which the 311th amino acid residue in the amino acid sequence shown in SEQ ID NO: 15 is substituted with an amino acid other than Thr. it can. A more preferred genetic engineering technique is a method of recombining the AK gene in the chromosome of the coryneform bacterium with the desensitized AK gene by homologous recombination. Furthermore, it is preferable to obtain the desensitized AK gene by a technique for introducing a desired mutation into the AK gene (for example, a method using a QuitChange site-directed mutagenesis kit manufactured by Stratagene).

  The fermentation raw material for the coryneform bacterium may be any material that promotes the growth of the coryneform bacterium to be fermented and cultivated and can satisfactorily produce a chemical product that is a target fermentation product. As a fermentation raw material, for example, a normal liquid medium that appropriately contains a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins is preferably used.

  Examples of the carbon source include saccharides such as glucose, fructose, sucrose, fructose, galactose and lactose, starch containing these saccharides, potato, wheat, corn, sweet potato, rice, cassava, tapioca, kudzu, katakuri, Hydrolyzed starch from green beans, sago palm, bracken, sugar cane molasses, sugar beet molasses, sugar beet, cane juice, sugar beet molasses or cane juice extract or concentrate, sugar beet molasses or cane juice filtrate, syrup (high test Molasses), raw sugar or raw sugar refined or crystallized from sugar beet molasses or cane juice, purified sugar crystallized or crystallized from canola juice or cane juice, organic acids such as acetic acid and fumaric acid, ethanol, etc. Alcohols and glycerin Etc. is used. Sugars are the first oxidation products of polyhydric alcohols, and are carbohydrates that have one aldehyde group or ketone group, sugars with aldehyde groups are classified as aldoses, and sugars with ketone groups as ketoses. Point to.

  The concentration of the carbon source used in the fermentation raw material used in the present invention is not particularly limited, and is preferably as high as possible as long as it does not inhibit the production of chemical products. Usually, the concentration of the carbon source is preferably 1 (w / v)% or more and 50 (w / v)% or less, more preferably 3 (w / v)% or more and 30 (w / v)% or less in the fermentation medium. It is. The carbon source can be used after being concentrated or diluted with water.

  Examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein decomposition products, Other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used.

  Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, sodium salt, iron salt, manganese salt etc. can be added and used suitably, for example.

  In addition, trace nutrient sources such as biotin, thiamine, and vitamin B6 can be added as necessary. These micronutrient sources can be substituted with medium additives such as meat extract, yeast extract, polypeptone, polypeptone S, corn steep liquor, casamino acid and the like.

  When the microorganisms or cultured cells used in the present invention require specific nutrients for growth, the nutrients can be added as preparations or natural products containing them. An antifoaming agent can be added and used as necessary.

  In the present invention, the fermentation culture liquid refers to a liquid obtained as a result of growth of coryneform bacteria on the fermentation raw material. The composition of the fermented raw material to be added can be appropriately changed from the fermented raw material composition at the start of the culture so that the productivity of the target chemical product is increased.

  There are no particular limitations on the culture conditions for coryneform bacteria, and the culture is performed under aerobic conditions such as shaking culture and deep aeration and agitation culture. There is no particular limitation on the culture form used for chemical productivity evaluation, and the culture is performed by test tube culture, flask culture, batch culture, fed-batch fermentation, continuous culture, or the like. The culture temperature is 25 to 42 ° C, preferably 28 to 38 ° C. The culture time is not particularly limited, and the usual batch culture is 1 to 9 days.

  For pH adjustment of coryneform bacteria, divalent ion hydroxides such as ammonia, calcium carbonate and calcium hydroxide, hydrochloric acid, sulfuric acid or dicarboxylic acid can be used. The culture pH is preferably controlled to pH 5.0 to 8.0, more preferably pH 5.5 to 7.5. The state of the neutralizing agent is not limited and can be used as a gas, liquid, solid, or aqueous solution, but is preferably an aqueous solution or a slurry.

  In the culture of the present invention, if it is necessary to increase the oxygen supply rate, oxygen is added to the air to keep the oxygen concentration preferably at 21% or higher, the fermentation broth is pressurized, the stirring rate is increased, or the aeration rate is increased. It is possible to use means such as raising the value. Conversely, if it is necessary to reduce the oxygen supply rate, it is also possible to supply a gas containing no oxygen such as carbon dioxide, nitrogen and argon mixed with air.

  In addition, the present invention is characterized in that a fermentation broth of coryneform bacteria having the ability to produce chemical products is filtered through a separation membrane.

  The separation membrane used in the present invention is less likely to be clogged by coryneform bacteria used for fermentation, and maintains a high exclusion rate and high water permeability for which bacterial cells and sludge do not leak. Therefore, it is necessary to have a performance in which the filtration performance is stably continued with high accuracy and reproducibility. Therefore, in the present invention, as a separation membrane having such properties, the lower limit of the average pore diameter is 0.01 μm or more, preferably 0.01 μm or more, more preferably 0.02 μm or more, further preferably 0.04 μm or more, A porous membrane having an upper limit of less than 1 μm, preferably 0.4 μm or less, more preferably 0.2 μm or less is used.

  The average pore diameter of the porous membrane is obtained by measuring and averaging the diameters of all the pores that can be observed within a range of 9.2 μm × 10.4 μm in a scanning electron microscope observation at a magnification of 10,000 times. Can be sought.

  The standard deviation σ of the average pore diameter of the porous membrane is preferably 0.1 μm or less. Furthermore, since the standard deviation of the average pore diameter is small, that is, when the pore diameters are uniform, a uniform permeate can be obtained and the fermentation operation management becomes easy. Smaller is preferable if it is smaller. The standard deviation σ of the average pore diameter is N (the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm), each measured diameter is Xk, and the average pore diameter is X (ave) It is calculated by the following (Formula 1).

In the porous membrane used in the present invention, the pure water permeability coefficient of the porous membrane before use can be used as an index of the permeability of the fermentation broth. The water permeability coefficient is calculated by measuring the water permeability at a head height of 1 m using purified water from a reverse osmosis membrane at a temperature of 25 ° C., and the pure water permeability coefficient of the porous membrane is 2 × 10 ⁻⁹ m ³ / is preferably m ² / s / pa or higher, pure water permeability coefficient is ^{2 × 10 -9 m 3 / m} 2 / s / pa or ^{6 × 10 -7 m 3 / m} 2 / s / pa or less If so, a practically sufficient amount of permeated water can be obtained.

  In addition, the membrane surface roughness of the porous membrane is a factor that affects the clogging of the porous membrane. By reducing the membrane surface roughness of the porous membrane, the clogging is suppressed and stable continuous fermentation is achieved. Is possible. In addition, by reducing the membrane surface roughness of the porous membrane, it can be expected that the shear force generated on the membrane surface will be reduced in the filtration of microorganisms, the destruction of microorganisms is suppressed, and the porous membrane is clogged. It is considered that stable filtration is possible for a long time by being suppressed. Specifically, if the membrane surface roughness is 0.1 μm or less, the peeling coefficient and membrane resistance of the porous membrane can be suitably reduced, and filtration can be carried out with a lower transmembrane pressure difference.

Here, the film surface roughness is an average value of the height in the direction perpendicular to the film surface, and can be measured using the following atomic force microscope (AFM) under the following conditions.
・ Atomic force microscope (Nanoscope IIIa manufactured by Digital Instruments)
・ Conditions Tip SiN cantilever (Digital Instruments)
Scan mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
Scanning range 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
Scanning resolution 512 × 512
-Sample preparation The membrane sample was immersed in ethanol at room temperature for 15 minutes, then immersed in RO water for 24 hours, washed, and then air-dried. The RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, to exclude impurities such as ions and salts. The pore size of the RO membrane is approximately 2 nm or less.

Membrane surface roughness _{d rough} from the Z-axis direction of the height of each point by the AFM, is calculated by the following equation (2).

  The structure of the porous membrane used as a separation membrane in the present invention will be described. The porous membrane has separation performance and water permeability according to the quality of the water to be treated and the application, and from the viewpoint of separation performance such as blocking performance, water permeability and dirt resistance, the porous membrane contains a porous resin layer. A membrane is preferred. Such a porous membrane has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. The porous substrate supports the porous resin layer and gives strength to the separation membrane.

  The material of the porous substrate is made of an organic material and / or an inorganic material, and organic fiber is preferably used among them. Preferred porous substrates are woven and non-woven fabrics using organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. Among them, the density control is relatively easy and the production is also easy. An easy and inexpensive nonwoven fabric is preferably used.

  Further, as described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be suitably used. Examples of the material of the organic polymer film include polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, polyolefin resin, Cellulose-based resins and cellulose triacetate-based resins may be used, and a mixture of resins mainly composed of these resins may be used. Here, the main component means that the component is contained in an amount of 50% by weight or more, preferably 60% by weight or more.

  Among them, the organic polymer membrane materials include polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethers that are easy to form in solution and have excellent physical durability and chemical resistance. A sulfone resin, a polyacrylonitrile resin, and a polyolefin resin are preferable, a polyvinylidene fluoride resin or a polyolefin resin is more preferable, and a polyvinylidene fluoride resin or a resin containing the same as the main component is most preferably used.

  Here, as the polyvinylidene fluoride-based resin, a homopolymer of vinylidene fluoride is preferably used. In addition to a homopolymer of vinylidene fluoride, a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is used. A polymer is also preferably used. Examples of vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trifluoride chloride.

  Examples of the polyolefin resin include polyethylene, polypropylene, chlorinated polyethylene, and chlorinated polypropylene, and chlorinated polyethylene is preferably used.

  The porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane. In the case of a flat membrane, the average thickness is selected according to the application, but is preferably 20 μm or more and 5000 μm or less, and more preferably 50 μm or more and 2000 μm or less.

  The separation membrane used in the present invention is preferably a porous membrane formed from a porous substrate and a porous resin layer. At that time, either the porous resin layer permeates the porous base material or the porous resin layer does not permeate the porous base material, and it is selected according to the application. The average thickness of the porous substrate is preferably selected in the range of 50 μm to 3000 μm.

  When the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably selected in the range of 200 μm to 5000 μm, and the film thickness is preferably selected in the range of 20 μm to 2000 μm. Further, a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.

  The porous membrane used in the present invention can be made into a separation membrane element by combining with a support. The form of the separation membrane element having a porous membrane is not particularly limited, but the separation membrane element in which a support plate is used as a support and the porous membrane used in the present invention is disposed on at least one side of the support plate is the present invention. It is one of the suitable forms of the separation membrane element which has a porous membrane used by 1). In this embodiment, when it is difficult to increase the membrane area, it is also a preferable aspect to dispose a porous membrane on both surfaces of the support plate in order to increase the water permeability.

  The porous membrane used in the present invention can be produced by the method for producing a porous membrane described in JP-A-2008-131931.

  In the present invention, the transmembrane pressure difference when the coryneform bacterium is filtered with a separation membrane may be any condition as long as the microorganisms and the medium components are not easily clogged, but the transmembrane pressure difference is 0.1 kPa or more and 20 kPa or less. Is preferably within the range of 0.1 kPa to 10 kPa, and more preferably within the range of 0.1 kPa to 5 kPa. If the range of the transmembrane pressure difference is exceeded, clogging of microorganisms and medium components may occur rapidly, leading to a decrease in the amount of permeated water, which may cause problems in the filtration operation.

  As a driving force for filtration, it is possible to generate a transmembrane pressure difference in the porous membrane by a siphon utilizing a liquid level difference (water head difference) of the fermentation broth and the porous membrane treated water. Moreover, a suction pump may be installed on the porous membrane treated water side as a driving force for filtration, or a pressure pump may be installed on the fermentation culture solution side of the porous membrane. The transmembrane pressure difference can be controlled by changing the liquid level difference between the fermentation broth and the porous membrane treated water, so there is no need to keep the inside of the fermentation reactor in a pressurized state. The ability to produce does not decrease. Moreover, since there is no need to keep the inside of the fermentation reaction tank in a pressurized state, a power means for circulating the fermentation broth between the membrane separation tank and the fermentation reaction tank becomes unnecessary, and a porous membrane is installed in the fermentation reaction tank. It is also possible.

  In addition, when a pump is used to generate the transmembrane pressure, the transmembrane pressure can be controlled by the suction pressure, and also by the gas or liquid pressure that introduces the pressure on the fermentation broth side. The transmembrane pressure can be controlled. When these pressure controls are performed, the pressure difference between the pressure on the fermentation broth side and the pressure on the porous membrane treated water side can be used as the transmembrane pressure difference, which can be used to control the transmembrane pressure difference.

  In the present invention, coryneform bacteria can be continuously fermented and cultured by holding or refluxing the unfiltered solution of the filtration treatment in the fermentation broth and continuously adding the fermentation raw material to the fermentation broth. . Continuous fermentation culture of coryneform bacteria using separation membranes can increase cell density compared to batch fermentation culture, so that the production rate can be improved and the improved production rate can be maintained for a long time. This is a preferred embodiment of the present invention.

  When carrying out continuous fermentation, the continuous fermentation method disclosed in WO2007 / 097260 is preferably employed. Continuous culture may be started after increasing the microorganism concentration by performing batch culture or fed-batch culture at the beginning of the culture, or continuous culture may be performed simultaneously with the start of culture by seeding a high concentration of cells. From an appropriate time, it is possible to supply fermentation raw materials, filter and extract microorganisms or cultured cells from the fermentation broth. The start timings of the fermentation raw material supply, filtration, and extraction of microorganisms or cultured cells from the fermentation broth do not necessarily have to be the same. Moreover, supply of fermentation raw materials, filtration, and extraction of microorganisms or cultured cells from the fermentation broth may be continuous or intermittent. Nutrients necessary for cell growth as described above may be added to the fermentation raw material so that the cell growth is continuously performed.

  Furthermore, the present invention is characterized in that a coryneform bacterium having the ability to produce a chemical product is cultured in a fermentation medium containing a cell wall synthesis inhibitor to obtain a fermentation broth containing the chemical product. The cell wall synthesis inhibitor herein refers to inhibiting or inhibiting the synthesis of mycolic acid, which is known as a component of the coryneform bacterium by the coryneform bacterium, or inhibiting the adhesion of mycolic acid to the cell wall. It is a drug that inhibits the synthesis of cell walls of coryneform bacteria, and the present inventor has shown that the filtration and separation performance of the resulting fermentation broth can be significantly improved by culturing while adding a cell wall synthesis inhibitor into the fermentation medium. I found it.

  The cell wall synthesis inhibitor added to the fermentation medium is not particularly limited, but mycolic acid synthesis inhibitors such as isonicotinic acid hydrazide (also called INH, isoniazid), drugs that inhibit mycolic acid adhesion to the cell wall such as ethambutol, pyrazine Specific examples include cell wall synthesis inhibitory antibiotics such as amide, rifampicin, etionamide, cerulenin, cycloserine or penicillin, Tween 40 or Tween 60, preferably a mycolic acid synthesis inhibitor or a drug that suppresses mycolic acid adhesion to the cell wall, More preferred is INH or ethambutol.

  Inhibitors of mycolic acid synthesis such as INH are known as therapeutic agents for tuberculosis, and are used to inhibit the synthesis of mycolic acid, which is a cell wall component of Gram-positive bacteria, which are coryneform bacteria. It is used to improve the conversion efficiency (Japanese Patent Laid-Open No. 2007-508840, J. BIOSCI. BIOENG. 92 (3), 201-213, 2001).

  Drugs that suppress mycolic acid adhesion to the cell wall, such as ethambutol, are also known as anti-tuberculosis drugs and are related to the biosynthesis of arabinan, which is a component of arabinogalactan on the cell wall, by inhibiting the synthesis of arabinan. Inhibits mycolic acid attachment to cell wall arabinogalactans by reducing mycolic acid accumulation (Biochimica et Biophysica Acta 1437, 325-332, 1999).

  The addition of the cell wall synthesis inhibitor to the fermentation medium is not particularly limited as long as it does not inhibit the production of chemicals by fermentation culture of coryneform bacteria, but the concentration of the cell wall synthesis inhibitor in the fermentation medium is 0.1 to 20 g / It is preferable to add so that it may become L.

  When using a mycolic acid synthesis inhibitor such as INH as a cell wall synthesis inhibitor, the concentration of the mycolic acid synthesis inhibitor in the fermentation medium is from the viewpoint of good growth of microorganisms, production of chemicals, and total cost. It is preferably 0.1 g / L or more, more preferably 0.3 g / L or more, and further preferably 0.5 g / L or more.

  When using a drug that suppresses mycolic acid adhesion to the cell wall, such as ethambutol, as the cell wall synthesis inhibitor, the concentration of the drug that suppresses mycolic acid adhesion to the cell wall in the fermentation medium should be From the viewpoint of product production and total cost, it is preferably 0.1 g / L or more, more preferably 0.2 g / L or more, and further preferably 0.3 g / L or more.

  Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of these examples.

[Analysis method of L-lysine and cadaverine concentration by HPLC]
・ Column used: CAPCELL PAK C18 (Shiseido)
Mobile phase 0.1% (w / w) phosphoric acid aqueous solution: acetonitrile = 4.5: 5.5
・ Detection UV360nm
Sample pretreatment 25 μl of analysis sample, 25 μl of 1,4-diaminobutane (0.03 M), 400 μl of sodium hydrogen carbonate (0.075 M) as an internal standard, and ethanol of 2,4-dinitrofluorobenzene (0.2 M) The solution is added and mixed and incubated at 37 ° C. for 1 hour. After dissolving 50 μl of the reaction solution in 1 ml acetonitrile, 10 μl of the supernatant after centrifugation at 10,000 rpm for 5 minutes was analyzed by HPLC.

[Analysis method of glucose concentration by HPLC]
-Column used Luna NH2 250 × 4.6 mm (Phenomenex)
Mobile phase Milli-Q water: acetonitrile = 25: 75
・ Detection RI (differential refractive index)
Sample pretreatment The collected sample was filtered through a 0.2 μm filter, and 10 μl was subjected to HPLC analysis.

[Reference Example 1] Preparation of Corynebacterium glutamicum having resistance to S- (2-aminoethyl) -L-cysteine Corynebacterium glutamicum ATCC13032 strain according to the method disclosed in Example 2 of JP-A-2004-22569 A desensitization type AK gene was introduced so that it could be expressed on the chromosome to produce a TR-AK strain. The AEC resistance of the TR-AK strain was confirmed by the AEC resistance confirmation method disclosed in Example 2 of Japanese Patent Application Laid-Open No. 2004-222569.

[Reference Example 2] Preparation of Corynebacterium glutamicum having L-lysine decarboxylation activity, lacking homoserine dehydrogenase activity, and having resistance to S- (2-aminoethyl) -L-cysteine The TR-CAD2 strain was prepared by the method disclosed in Example 1 and Example 2 of No. 222569.

[Example 1 and Comparative Example 1] Production of chemicals using TR-AK strain TR-AK strain (Example 1 and Comparative Example 1) was sterilized LB medium (chloramphenicol (10 μg / ml) added) 10 ml One platinum ear was inoculated to each other and shaken at 30 ° C. for 24 hours and cultured in advance. The pre-cultured solution was inoculated in 100 ml of the same medium as the pre-culture, and cultured for 24 hours under conditions of 30 ° C., amplitude 30 cm and 180 rpm. Next, 3900 ml of the fermentation medium shown in Table 1 was inoculated with the whole preculture solution, and cultured for 60 hours while adjusting the pH to 7 at 30 ° C. with a stirring blade speed of 800 rpm while aerating sterilized air at 0.5 vvm. Went. In Example 1, 1 g / L INH was added to the fermentation medium shown in Table 1 (hereinafter referred to as MI1), and Comparative Example 1 was not added INH to the fermentation medium shown in Table 1. The neutralization was carried out with aqueous hydrochloric acid (3M) and aqueous ammonia (3M). After completion of the culture, a part of the culture solution was taken out and centrifuged at 4 ° C. and 8,000 rpm for 10 minutes to remove the cells and collect the culture supernatant. L-lysine and cadaverine in the culture supernatant were analyzed by HPLC. In Example 1, the L-lysine concentration was about 9.4 g / L, and the cadaverine concentration was about 0 g / L. In Comparative Example 1, the L-lysine concentration was about 9.4 g / L, and the cadaverine concentration was about 0 g / L. Table 2 shows the results of examining the fastest filtration time using the fermentation broth after completion of fermentation in Example 1 and Comparative Example 1. FIG. 1 is a graph comparing the filtration time and the transmembrane pressure when filtration is performed with an equivalent filtration flux using the fermentation broth after completion of fermentation in Example 1 and Comparative Example 1. From Table 2 and FIG. 1, according to the chemical production method of the present invention, the addition of INH has the effect of making it difficult to clog the separation membrane, and the filterability of the membrane is improved, so the filtration time is shortened or the amount of filtration is increased. It has become clear that the productivity of chemicals by means of is improved.

[Example 2 and Comparative Example 2] Production of chemicals using TR-AK strain TR-AK strain (Example 2 and Comparative Example 2) was sterilized LB medium (chloramphenicol (10 µg / ml) added) 10 ml. One platinum ear was inoculated, and the mixture was shaken at 30 ° C. for 24 hours and cultured in advance. The pre-culture solution was inoculated in 100 ml of the same medium as the pre-culture, and cultured for 24 hours under conditions of 30 ° C., amplitude 30 cm and 180 rpm. Next, 3900 ml of the fermentation medium shown in Table 1 was inoculated with the whole preculture solution, and cultured for 60 hours while adjusting the pH to 7 at 30 ° C. with a stirring blade speed of 800 rpm while aerating sterilized air at 0.5 vvm. Went. In Example 2, 5 g / L of INH was added to the fermentation medium shown in Table 1 (hereinafter referred to as MI5), and in Comparative Example 2, the antifoaming agent PE-L was used for foam suppression without adding INH to the fermentation medium shown in Table 1. Was added at 0.05%. The neutralization was carried out with aqueous hydrochloric acid (3M) and aqueous ammonia (3M). After completion of the culture, a part of the culture solution was taken out and centrifuged at 4 ° C. and 8,000 rpm for 10 minutes to remove the cells and collect the culture supernatant. L-lysine and cadaverine in the culture supernatant were analyzed by HPLC. In Example 2, the L-lysine concentration was about 9.4 g / L, and the cadaverine concentration was about 0 g / L. In Comparative Example 2, the L-lysine concentration was about 9.4 g / L, and the cadaverine concentration was about 0 g / L. Table 2 shows the results of examining the fastest filtration time using the fermentation broth after completion of fermentation in Example 2 and Comparative Example 2. FIG. 2 is a graph comparing the filtration time and the transmembrane pressure when filtration is performed with an equivalent filtration flux using the fermentation broth after completion of fermentation in Example 2 and Comparative Example 2. From Table 2 and FIG. 2, according to the method for producing a chemical product of the present invention, the addition of INH has the effect of making it difficult to clog the separation membrane, and the filterability of the membrane is improved. It became clear that the sex was improved.

[Example 3, Comparative Example 3] Production of chemicals using TR-CAD2 strain TR-CAD2 strain (Example 3, Comparative Example 3) was sterilized LB medium (chloramphenicol (10 μg / ml) added) 10 ml One platinum ear was inoculated, and the mixture was shaken at 30 ° C. for 24 hours and cultured in advance. The pre-culture solution was inoculated in 100 ml of the same medium as the pre-culture, and cultured for 24 hours under conditions of 30 ° C., amplitude 30 cm and 180 rpm. Next, 3900 ml of the fermentation medium shown in Table 1 (in Example 3, 10 g / L of INH was added to the fermentation medium of Table 1 (hereinafter referred to as MI10), and Comparative Example 3 was not added with INH) was planted with the total amount of the preculture. Culturing was carried out for 60 hours while adjusting the pH to 7 at 30 ° C. with a stirring blade rotating at 800 rpm while aerated and sterilized air was vented at 0.5 vvm. The neutralization was carried out with aqueous hydrochloric acid (3M) and aqueous ammonia (3M). After completion of the culture, a part of the culture solution was taken out and centrifuged at 4 ° C. and 8,000 rpm for 10 minutes to remove the cells and collect the culture supernatant. L-lysine and cadaverine in the culture supernatant were analyzed by HPLC. In Example 3, the L-lysine concentration was about 0 g / L, and the cadaverine concentration was about 9.4 g / L. In Comparative Example 3, the L-lysine concentration was about 0 g / L, and the cadaverine concentration was about 9.4 g / L. Table 2 shows the results of examining the fastest filtration time using the fermentation broth after completion of fermentation in Example 3 and Comparative Example 3. FIG. 3 is a graph comparing the filtration time and the transmembrane pressure difference when filtration is performed with an equivalent filtration flux using the fermented liquids after completion of fermentation in Example 3 and Comparative Example 3. From Table 2 and FIG. 3, according to the method for producing a chemical product of the present invention, the addition of INH has the effect of making the separation membrane difficult to clog, and the filterability of the membrane is improved. It became clear that the sex was improved.

[Example 4, Comparative Example 4] Production of chemicals using TR-CAD2 strain TR-CAD2 strain (Example 4, Comparative Example 4) was sterilized LB medium (chloramphenicol (10 μg / ml) added) 10 ml One platinum ear was inoculated, and the mixture was shaken at 30 ° C. for 24 hours and cultured in advance. The pre-culture solution was inoculated in 100 ml of the same medium as the pre-culture, and cultured for 24 hours under conditions of 30 ° C., amplitude 30 cm and 180 rpm. Next, 3900 ml of the fermentation medium shown in Table 1 (in Example 4, 5 g / L of ethambutol was added to the fermentation medium of Table 1 (hereinafter referred to as ET5), and in Comparative Example 4, no ethambutol was added) was planted with the total amount of the preculture. Culturing was carried out for 60 hours while adjusting the pH to 7 at 30 ° C. with a stirring blade rotating at 800 rpm while aerated and sterilized air was vented at 0.5 vvm. The neutralization was carried out with aqueous hydrochloric acid (3M) and aqueous ammonia (3M). After completion of the culture, a part of the culture solution was taken out and centrifuged at 4 ° C. and 8,000 rpm for 10 minutes to remove the cells and collect the culture supernatant. L-lysine and cadaverine in the culture supernatant were analyzed by HPLC. In Example 4, the L-lysine concentration was about 0 g / L, and the cadaverine concentration was about 9.4 g / L. In Comparative Example 4, the L-lysine concentration was about 0 g / L, and the cadaverine concentration was about 9.4 g / L. Table 2 shows the results of examining the fastest filtration time using the fermentation broth after completion of fermentation in Example 4 and Comparative Example 4. FIG. 4 is a graph comparing the filtration time and the transmembrane differential pressure when the fermentation liquid after completion of fermentation in Example 4 and Comparative Example 4 was used and filtration was performed with an equivalent filtration flux. From Table 2 and FIG. 4, according to the method for producing a chemical product of the present invention, addition of ethambutol has the effect of making the separation membrane difficult to clog, and the filterability of the membrane is improved. It became clear that the sex was improved.

[Example 5, Comparative Example 5] Continuous production of chemicals using TR-AK strain Whether or not L-lysine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in Fig. 1 of WO2007 / 097260. Therefore, a continuous fermentation test using the same apparatus was conducted. Fermentation medium (MI5 medium in Example 5 and medium shown in Table 1 in Comparative Example 4) was used after high-pressure steam sterilization at 121 ° C. for 15 minutes. The separation membrane element adopts the form shown in FIG. 3 posted in WO2007 / 097260, and the separation membrane is a porous membrane mainly composed of polyvinylidene fluoride (PVDF) produced according to Reference Example 2 of WO2007 / 097260. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. When the characteristics of the porous membrane were examined, the average pore diameter was 0.1 μm, and the pure water permeability coefficient was 50 × 10 ⁻⁹ m ³ / m ² / s / pa. The operating conditions in Example 5 and Comparative Example 5 are as follows unless otherwise specified.

[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element effective filtration area: 120 cm ²
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.5 vvm
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: Incubation tank containing separation membrane element, and all used media are autoclaved at 121 ° C. for 20 minutes ・ High-pressure steam sterilization ・ pH adjustment: pH adjusted to 7.0 with aqueous hydrochloric acid (3M) and aqueous ammonia (3M) Adjustment / Membrane permeate flow rate control: Flow rate was controlled by the difference in the water head of the membrane separation tank (the water head difference was controlled within 2 m).

  TR-AK strain (Example 5, Comparative Example 5) was inoculated with 1 platinum ear in 10 ml of sterilized LB medium (chloramphenicol (10 μg / ml) added) and shaken at 30 ° C. for 24 hours. Culture was performed. This pre-culture medium was inoculated in 100 ml of the same medium as the pre-culture, and cultured for 24 hours under conditions of 30 ° C., amplitude 30 cm and 180 rpm. 1 L fermentation medium (MI5 medium in Example 5 and Comparative Example 5 in Example 5) was passed through the culture medium aerated at 0.5 vvm through the sterilized air of the continuous fermentation apparatus shown in FIG. 1 of WO2007 / 097260. The culture medium shown in Table 1) is inoculated, the fermentation reaction tank 1 is stirred at 800 rpm with the attached stirrer 5, the temperature of the fermentation reaction tank 1 is adjusted to the temperature of 30 ° C., and the culture is performed for 24 hours. Performed (preculture). Immediately after completion of the pre-culture, the MI5 medium (Example 5) or the medium shown in Table 1 (Comparative Example 5) is continuously supplied, and the amount of membrane permeate is adjusted so that the amount of fermentation broth in the continuous fermentation apparatus becomes 1.5 L. Continuous culture was performed while controlling, and L-lysine was produced by continuous fermentation at 30 ° C. At this time, sterilized air was supplied into the fermentation reaction tank 1 from the gas supply device. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. The produced L-lysine concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately.

  As a result, the continuous fermentation for 220 hours in Comparative Example 5 was continued for 340 hours in Example 5 (FIG. 5), and the membrane filterability was improved by adding INH to the fermentation medium for a long time. Continuous fermentation became possible, and the productivity of L-lysine was improved (Table 3).

[Example 6, Comparative Example 6] Continuous production of chemicals using TR-CAD2 strain In order to examine whether cadaverine can be obtained by continuous fermentation by operating the continuous fermentation apparatus shown in Fig. 2 of WO2007 / 097260 Then, a continuous fermentation test using the same apparatus was performed. Fermentation medium (MI1 medium for Example 6 and medium shown in Table 1 for Comparative Example 6) was used after high-pressure steam sterilization at 121 ° C. for 15 minutes. As the separation membrane and separation membrane element, those of Example 5 and Comparative Example 5 were used. The operating conditions in Example 6 and Comparative Example 6 are as follows unless otherwise specified.

[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element effective filtration area: 120 cm ²
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.5 vvm
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: Incubation tank containing separation membrane element, and all used media are autoclaved at 121 ° C. for 20 minutes ・ High-pressure steam sterilization ・ pH adjustment: pH adjusted to 7.0 with aqueous hydrochloric acid (3M) and aqueous ammonia (3M) Adjustment / Membrane permeate flow rate control: Flow rate was controlled by the difference in the water head of the membrane separation tank (the water head difference was controlled within 2 m).

  First, TR-CAD2 strain (Example 6, Comparative Example 6) was inoculated with 1 platinum ear in 10 ml of sterilized LB medium (chloramphenicol (10 μg / ml) added) and shaken at 30 ° C. for 24 hours. Each culture was performed. This pre-culture medium was inoculated in 100 ml of the same medium as the pre-culture, and cultured for 24 hours under conditions of 30 ° C., amplitude 30 cm and 180 rpm. 2 L of fermentation medium (MI1 medium for Example 6 and Table 1 for Comparative Example 6) was passed through the culture medium aerated at 0.5 vvm through the sterilized air of the continuous fermentation apparatus shown in FIG. 2 of WO2007 / 097260. 1), the fermentation reaction tank 1 is stirred at 800 rpm with the attached stirrer 5, the aeration rate of the fermentation reaction tank 1 is adjusted and the temperature is adjusted to 30 ° C., and the culture is performed for 24 hours. (Pre-culture). Immediately after completion of the preculture, the MI1 medium (Example 6) or the medium shown in Table 1 (Comparative Example 6) is continuously supplied, and the amount of membrane permeate is adjusted so that the fermentation broth of the continuous fermentation apparatus becomes 1.5 L. Continuous culture was performed while controlling, and cadaverine was produced by continuous fermentation at 30 ° C. At this time, sterilized air was supplied into the fermentation reaction tank 1 from the gas supply device. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. The produced cadaverine concentration and residual glucose concentration in the membrane permeation fermentation broth were measured as appropriate.

  As a result, compared with the continuous fermentation for 200 hours in Comparative Example 6, the fermentation in Example 6 was continuous for 320 hours (FIG. 6), and the filterability of the membrane was improved by adding INH to the fermentation medium. Continuous fermentation became possible and cadaverine productivity was improved (Table 3).

[Example 7, Comparative Example 7] Continuous production of chemicals using TR-CAD2 strain Polyvinylidene fluoride prepared according to Reference Example 13 of WO2007 / 097260 as a separation membrane instead of the porous membrane used in Example 5 A hollow fiber membrane mainly composed of (PVDF) was used. As the separation membrane element, the form shown in FIG. 4 of WO2007 / 097260 was adopted. A continuous fermentation test was conducted in the same manner as in Example 6 except that the MI5 medium for Example 7 was used. As a result, the continuous fermentation for 210 hours in Comparative Example 7 was continued for 330 hours in Example 7 (FIG. 7), and the filterability of the membrane was improved by adding INH to the fermentation medium. Continuous fermentation was possible, and cadaverine productivity was improved (Table 3).

[Example 8, Comparative Example 8] Continuous production of chemicals using TR-CAD2 strain Polyvinylidene fluoride prepared according to Reference Example 13 of WO2007 / 097260 as a separation membrane instead of the porous membrane used in Example 5 A hollow fiber membrane mainly composed of (PVDF) was used. As the separation membrane element, the form shown in FIG. 4 of WO2007 / 097260 was adopted. A continuous fermentation test was conducted in the same manner as in Example 5 except that the ET5 medium for Example 8 was used. As a result, the continuous fermentation for 220 hours in Comparative Example 8 was continued for 340 hours in Example 7 (FIG. 8), and the filterability of the membrane was improved by adding ethambutol to the fermentation medium. Continuous fermentation was possible, and cadaverine productivity was improved (Table 3).

Claims (7)

Coryneform bacteria that have the ability to produce chemicals are cultured in a fermentation medium containing a mycolic acid synthesis inhibitor or a drug that suppresses mycolic acid adhesion to the cell wall, and the average pore size is determined using the resulting fermentation broth as a separation membrane. A method for producing a chemical product, characterized by performing filtration using a porous membrane having pores of 0.01 μm or more and less than 1 μm and recovering a product from the filtrate. The method for producing a chemical product according to claim 1, wherein the mycolic acid synthesis inhibitor or the drug that suppresses mycolic acid adhesion to a cell wall is isonicotinic acid hydrazide (INH) or ethambutol. The chemical product according to claim 1 or 2 , wherein the content of the mycolic acid synthesis inhibitor in the fermentation medium or the drug that suppresses mycolic acid adhesion to the cell wall is 0.1 to 20 g / L. Manufacturing method. The culture of the coryneform bacterium is a continuous fermentation culture in which the unfiltered liquid of the filtration treatment is retained or refluxed in the fermentation culture liquid and the fermentation medium is continuously added. The manufacturing method of the chemical in any one of 1-3 . The method for producing a chemical product according to any one of claims 1 to 4 , wherein the coryneform bacterium is a genus Corynebacterium or Brevibacterium. The method for producing a chemical product according to any one of claims 1 to 5 , wherein the chemical product is an amino acid or a polyamine. The chemical is a L- lysine or cadaverine, chemical manufacturing method according to any one of claims 1-6.

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