WO2001005979A1

WO2001005979A1 - Process for producing target substance by fermentation method

Info

Publication number: WO2001005979A1
Application number: PCT/JP2000/004774
Authority: WO
Inventors: Eiichiro Kimura; Hisao Ito; Osamu Kurahashi
Original assignee: Ajinomoto Co., Inc.
Priority date: 1999-07-19
Filing date: 2000-07-14
Publication date: 2001-01-25
Also published as: JP2003204783A; AU6018400A

Abstract

A process for producing a target substance by using a microorganism which comprises culturing the microorganism in a medium, producing and accumulating the target substance in the medium, and then collecting the target substance, wherein use is made of a microorganism capable of producing the target substance and having an enhanced RNA polymerase activity as the microorganism to thereby improve the productivity of the target substance.

Description

TECHNICAL FIELD The present invention relates to a method for producing a target substance using a microorganism, and more particularly, to a method for producing a target substance such as L-amino acid, an antibiotic, a vitamin, a growth factor, or a physiologically active substance. It discloses a means for improving the productivity of a target substance in a method of producing using a microorganism. BACKGROUND ART As a typical method for producing a substance using a microorganism, a method for producing L-amino acid by a fermentation method is known. L-amino acid is used not only as a seasoning and food, but also as a component of various nutrient mixtures for medical purposes. In addition, it is used as an animal feed additive, as a reagent in the pharmaceutical and chemical industries, and as a growth factor for the production of L-amino acids such as L-lysine and L-homoserine by microorganisms. Known microorganisms capable of producing L-amino acids by fermentation include coryneform bacteria, Escherichia bacteria, Bacillus bacteria, and Serratia bacteria.

To produce L-amino acids by fermentation, a method using a wild-type microorganism (wild strain), a method using an auxotroph derived from the wild strain, and various drug-resistant mutants derived from the wild strain There are methods using metabolic regulatory mutants, and methods using strains having both auxotrophic and metabolic regulatory mutants.

In recent years, recombinant DNA technology has been used for fermentative production of L-amino acids. In this technology, the principle is to enhance the L-amino acid biosynthesis system of the host microorganism by enhancing the gene encoding the L-amino acid biosynthesis enzyme. For more information on these circumstances, see, for example,

1986 ”.

In addition to L-amino acids, many substances are produced by fermentation using microorganisms. No. For example, antibiotics and vitamins are examples. In the fermentative production of these substances, the use of recombinant DNA technology mainly involves enhancement of genes encoding biosynthetic enzymes of the target substance or its precursor.

The productivity of target substances has been remarkably improved by the above-mentioned microorganism breeding technology.

On the other hand, substances produced by microorganisms are affected by various biochemical reactions in addition to the biosynthetic system of the substance, and are affected by the growth of microorganisms. Therefore, in order to improve the production efficiency of the target substance, various studies on culture conditions such as a culture medium and a culture method have been conducted. However, the relationship between the RNA polymerase activity and the growth of microorganisms and the productivity of the target substance has not been studied. DISCLOSURE OF THE INVENTION The present invention relates to a method for producing a target substance such as an L-amino acid, an antibiotic, a vitamin, a growth factor, or a physiologically active substance using a microorganism. It is an object to provide a method for improvement.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that when the RNA polymerase activity of a microorganism is enhanced, the growth of the microorganism is improved and the production amount of a target substance is increased. The invention has been completed.

That is, the present invention is as follows.

(1) A microorganism capable of producing a target substance and having enhanced RNA polymerase activity.

(2) The microorganism according to (1), wherein the target substance is an L-amino acid.

(3) The microorganism of (1), wherein the RNA polymerase activity is enhanced by increasing the copy number of each of the rpoA, rpoB, 00, and poD genes.

(4) The microorganism according to (1), wherein the microorganism is a bacterium belonging to the genus Escherichia or a coryneform bacterium.

(5) A method comprising culturing the microorganism according to any of (1) to (4) in a medium, producing and accumulating a target substance in the culture, and collecting the target substance from the culture. Method of manufacturing a material.

As used herein, the term “target substance producing ability” refers to the microorganism of the present invention in a medium. The ability to accumulate a significant amount of a target substance in a medium or cells when cultured. Hereinafter, the present invention will be described in detail.

The target substance produced by the present invention is not particularly limited as long as it can be produced by a microorganism. For example, L-threonine, L-lysine, L-glutamic acid, L-one-isine, L-isoleucine, L-valine, L- Various L-amino acids such as phenylalanine are exemplified. In addition, substances that are biosynthesized by microorganisms, such as nucleic acids such as guanylic acid and inosinic acid, vitamins, antibiotics, growth factors, and bioactive substances. In addition, it goes without saying that the present invention can be used for substances that are not currently produced using microorganisms, as long as they can be produced by microorganisms.

The microorganism used in the present invention is not particularly limited, and any microorganism conventionally used for producing a useful substance by a fermentation method can be used. In addition, the present invention can be applied to microorganisms that have not been conventionally used industrially as long as they have the ability to produce the target substance. The microorganism of the present invention may originally have the ability to produce the target substance, or may be given the ability to produce the target substance by breeding using a mutation method or recombinant DNA technology. It may be something.

Specific examples include Escherichia coli bacteria such as Escherichia coli, coryneform bacteria such as Brevibacterium lactofermenum, Bacillus bacteria such as Bacillus subtilis, and Serratia bacteria such as Serratia marcescens. , But not limited to these.

More specifically, the following strains can be mentioned. For example, when the target substance is L-threonine, Escherichia coli VKPM B-3996 (RIA 1867) (see US Pat. No. 5,175,107), Corynebacterium acetoacidophilum AJ12318 (FERM BP-1172) (See U.S. Pat. No. 5,188,949), and in the case of L-lysine, Escherichia coli AJ11442 (NRRL B-12185, FERM BP-1543) (see U.S. Pat. No. 4,346,170), Brevibacte Lium lactofermentum AJ11082 (NRRL B-11470), Brevibacterium lactofamentum AJ3990 1 ¹ 31269) (see U.S. Pat. No. 4,066,501) and the like in the case of L-glutamic acid. '' Kori AJ12624 (FERM BP-3853) (See French Patent Application Publication No. 2,680,178), Brevipacterium. Lactofa-mentum AJ12475 (FERM BP-2922) (see US Pat. No. 5,272,067), and L—leucine for Essieri. Here. AJ11478 (FERM P-5274) (see Japanese Patent Publication No. 62-34397), Brevipacterium 'Lactofermentum AJ3718 (FERM P-2516) (see US Patent No. 3,970,519), etc. In the case of L-isoleucine, Escherichia coli KX141 (VKPM B-4781) (see EP-A-519,113), Brevibacterium flavum AJ12149 (FERM BP-759) (US patent) No. 4, 656, 135), and in the case of L-palin, Escherichia coli VL1970 (VKPM B-4411)) (see European Patent Application Publication No. 519, 113), Brevipacterium 'Lactov Amentham AJ12341 (FERM BP-1763) (see US Pat. No. 5,188,948) and the like. In the case of L-phenylalanine, Escherichia coli AJ12604 (FERM BP-3579) (see European Patent Application Publication No. 488,424), Brevipacterium lactofermentum AJ12637 (FERM BP-4160) (See French Patent Application Publication No. 2,686,898).

The microorganism used in the present invention is a microorganism having an ability to produce a target substance and having enhanced RNA polymerase activity. RNA polymerase is composed of the subunits of HI, β,? ', And HI, and HI, β, and β' are encoded by rpoA, rpoB, and rpoC genes, respectively. In Escherichia coli, rpo B and rpo C form an operon. Also, there are a plurality of types of σ subunits, and in Escherichia coli, ³² and ⁷² are known, each of which is coded by rpoH and rpoD. Of these, rp0D functions specifically during the growth phase and is preferred in the present invention.

To enhance RNA polymerase activity, a gene fragment encoding each RNA polymerase subunit is ligated to a vector, preferably a multicopy vector, that functions in the target microorganism to produce recombinant DNA. Then, it may be introduced into a target microorganism and transformed. Increased intracellular copy number of each gene results in enhanced RNA polymerase activity.

As a gene encoding each subunit of RNA polymerase, a gene of any microorganism such as a bacterium belonging to the genus Escherichia such as Escherichia coli or a coryneform bacterium can be used. In Escherichia coli, rp oA, rp o B, rp o C, Since the nucleotide sequence of each gene of rpo D has been elucidated (rp0A: GenBank / EMBL / DDBJ Accession J01685, rpo B, rpo C: GenBank / EMBL / DDBJ Accession J01678, rpo D: GenBank / EMBL / DDBJ Accession J01687), a primer can be synthesized based on these nucleotide sequences, and the chromosomal DNA of Escherichia coli can be transformed into a type II and obtained by PCR.

The primers shown in SEQ ID NOs: 1 and 2 were used as primers for amplifying the rp0A gene, the primers shown in SEQ ID NOS: 3 and 4 were used as primers for amplifying the rpo BC op Primers for amplification include the primers shown in SEQ ID NOS: 5 and 6.

Each gene amplified by the PCR method is connected to a vector DNA capable of autonomous replication in cells such as Escherichia coli 'corynecoryneform bacteria' to prepare recombinant DNA, which is then introduced into Escherichia coli cells. , The subsequent operations become slow. As a vector capable of autonomous replication in Escherichia coli cells, a brassmid vector is preferable, and a vector capable of autonomous replication in host cells is preferable.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010, etc. Is mentioned. Examples of vectors capable of autonomous replication in coryneform bacterium cells include pAM330 (see Japanese Patent Application Publication No. 58-67699), pHM1519 (see Japanese Patent Application Publication No. 58-77895), and the like. In addition, a DNA fragment having the ability to enable autonomous replication of plasmid in coryneform bacteria is extracted from these vectors and inserted into the Escherichia coli vector, whereby autonomous replication in both Escherichia coli and coryneform bacteria occurs. Not possible Can be used as a shuttle vector.

Such shuttle vectors include the following. Microorganisms carrying the respective vectors and the accession numbers of the international depository organizations are shown in parentheses.

PAJ655 Escherichia ·] Re AJ11882 (FERM BP-136)

Coryneha, Cterium-ku, Rutamicum SR8201 (ATCC39135)

PAJ1844 Escherichia 'Kori AJ11883 (FERM BP-137)

Coryneha ,, ゥ, ', ルミ 820 SR8202 (ATCC39136)

PAJ611 Escherichia Cori AJ11884 (FERM BP-138)

PAJ3148 Coryneha, Cterizum, Rutamicum SR8203 (ATCC39137) PAJ440 A ^ X- ^ 7 ^ 'JXAJ11901 (FERM BP-140)

pHC4 Escherichia coli AJ12617 (FERM BP-3532)

These vectors are obtained from the deposited microorganism as follows. The cells collected during the logarithmic growth phase were lysed using lysozyme and SDS, centrifuged at 300,000 X g, and polyethylene glycol was added to the supernatant obtained from the lysate. Separate and purify the mixture by centrifugation at the equilibrium density gradient of ethidium.

In order to prepare a recombinant DNA by ligating a gene encoding each subunit of RNA polymerase and a vector, the vector is cut with a restriction enzyme that matches the end of the DNA fragment containing each gene. The DNA fragment and the vector are ligated. Ligation is usually performed using a ligase such as T4DNA ligase. All of the genes encoding each subunit may be inserted into a single vector, or may be separately inserted into two or three or more different vectors. In the examples described below, two types of recombinant vectors, one obtained by inserting the rpoA gene and the rpo BC operon into the same vector and the other obtained by inserting the rpoD gene into another vector, were used. Each of these genes was introduced into a coryneform bacterium by using a recombinant vector.

Transformation by introducing the recombinant DNA prepared as described above into a microorganism may be performed according to a transformation method according to the microorganism to be used which has been reported so far. For example, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and a method for preparing DNA from transgenic cells and introducing DNA as described in Bacillus' subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (19 77)). Alternatively, the recombinant DNA is introduced into the DNA recipient by transforming the cells of the DNA recipient into protoplasts or spheroplasts that readily incorporate the recombinant DNA, as is known for Bacillus subtilis, actinomycetes, and yeast. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JM and Hopwood, 0. A., Nature, 274, 398 (1978); Hin Thigh, A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)) are also applicable. For the coryneform bacterium, the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) is available. It is effective.

Enhancement of RNA polymerase activity can also be achieved by allowing multiple copies of the gene encoding each of the RNA polymerase subunits to be present on the chromosome DNA of the microorganism. In order to introduce multiple copies of a DNA fragment on the chromosome DNA of a microorganism, homologous recombination is performed using a sequence present on the chromosome DNA in multiple copies as a target. Sequences present in multiple copies on the chromosome DNA include native DNA and inverted repeats present at the end of the transposable element. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, a gene encoding each of the RNA polymerase subunits may be mounted on a transposon and transferred to introduce multiple copies into chromosomal DNA. It is possible. Either method results in an increase in the copy number of the gene encoding each of the RNA polymerase subunits in the transformant, resulting in an increase in RNA polymerase activity.

In addition to the above-described gene amplification, the enhancement of RNA polymerase activity can also be achieved by replacing the expression regulatory sequence such as the promoter of the gene encoding each of the subunits of RNA polymerase with a strong one. Is achieved (see Japanese Patent Application Laid-Open No. 1-215280). For example, lac promoter evening one, trp promoter, trc promoter evening one, tac promoter, P _R promoter of lambda phage, P _L promoter, tet promoter evening one, amyE promoter evening one, spac promoter and so forth are known as strong promoters I have. Replacement with these promoters enhances RNA polymerase activity by enhancing expression of genes encoding each of the RNA polymerase subunits. The enhancement of expression control sequences may be combined with increasing the copy number of each gene.

As long as the effects of the present invention are not impaired, the microorganism of the present invention has other properties such as enhanced RNA polymerase activity and enhanced biosynthetic enzymes of the target substance. It may be provided. As the biosynthetic enzymes of the target substance, for example, when the target substance is L-glutamic acid, glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isoquenate dehydrogenase, Aconitate hydrase, citrate synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, enolase, phosphogly Cellom enzyme, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosulfose isomerase, fructose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase, etc. is there. The microorganism of the present invention may have a reduced or defective activity of an enzyme that catalyzes a reaction that produces a compound other than the target substance by branching off from the target substance biosynthetic pathway. For example, when the target substance is L-glutamic acid, the enzyme may be a-ketoglutarate dehydrogenase, isoquenate lyase, acetyl phosphate transferase, acetate kinase, acetate hydroxy acid synthase Acetolactate synthase, acetyl formate transferase, lactate dehydrogenase, L-glutamate decarboxylase, 1-pyrroline dehydrogenase and the like. Furthermore, the microorganism of the present invention may be provided with other properties that are favorable for production of the target substance. For example, when the target substance is L-glutamic acid and the microorganism is a coryneform bacterium, a temperature-sensitive mutation to a biotin-inhibiting substance such as a surfactant is imparted to a medium containing an excessive amount of biotin. Thus, L-glutamic acid can be produced in the absence of a biotin action inhibitor (see W096 / 06180). Examples of such coryneform bacteria include Brevibacterium lactofermenum AJ13029 described in W096 / 06180. The AJ13029 strain was submitted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (Postal Code 305-8566, Tsukuba 1-3-1-3, Ibaraki, Japan) on September 2, 1994 under the accession number FERM P-14501. Deposited and transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given accession number FE RM BP-5189.

In addition, a coryneform bacterium capable of producing L-lysine and L-glutamic acid is subjected to a temperature-sensitive mutation against a biotin-inhibiting substance, so that the biotin-inhibiting substance can be expressed in a medium containing an excessive amount of biotin. L-Lysine and L-glutamic acid can be produced simultaneously in the absence (see W096 / 06180). Examples of such strains include Brevipacterium lactofermentum AJ12993 strain described in W096 / 06180. The strain was deposited on June 3, 1994 with the Institute of Life Science and Industrial Technology, National Institute of Advanced Industrial Science and Technology (zip code 305-8566, 1-3 1-3 Tsukuba, Higashi, Ibaraki, Japan) under the accession number FERM P-14348. It was transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given the accession number FERM BP-5188. Preparation of chromosomal DNA required for construction of the microorganism of the present invention, ligation of gene fragment to plasmid, PCR, preparation of plasmid DNA, DNA cleavage and ligation, transformation, setting of oligonucleotides used as primers, etc. Can employ ordinary methods well known to those skilled in the art. These methods are described in Sambrook, J., Friisch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manua 1, Second Edition", Cold Spring Harbor Laboratory Press, (1989). It is self-published.

The target substance is produced by culturing a microorganism having an improved target substance production ability in a medium as described above, producing and accumulating the target substance in the medium, and collecting the target substance from the culture. be able to.

As the medium, a well-known medium conventionally used depending on the microorganism to be used may be used. That is, it is a normal medium containing a carbon source, a nitrogen source, inorganic ions and other organic components as required. A special medium for carrying out the present invention is not particularly required.

As the carbon source, sugars such as glucose, lactose, galactose, hydrolysates of fructose and starch, alcohols such as glycerol and sorbitol, and organic acids such as fumaric acid, citric acid, and succinic acid are used. be able to. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, ammonia water and the like can be used.

As organic trace nutrients, it is desirable to include required substances such as vitamin B1, L-homoserine, and L-tyrosine, or an appropriate amount of yeast extract and the like. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. are added as needed.

The cultivation may be performed under well-known conditions conventionally used depending on the microorganism to be used. For example, culture is preferably performed under aerobic conditions for 16 to 120 hours, and the culture temperature is controlled at 25 ° C to 45 ° C, and the pH is controlled at 5 to 8 during the culture. For pH adjustment, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used. No special method is required in the present invention for collecting the target substance from the medium after the culture. That is, the present invention can be carried out by combining conventionally known ion exchange resin methods, precipitation methods and other methods. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to examples.

<1> Isolation of RNA polymerase gene from Escherichia coli and preparation of plasmid for RNA polymerase gene transfer

Whole genome DNA of E. coli W3110 strain was prepared by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)). Using this genomic DNA as type III, PCR was used to amplify the gene encoding each of the RNA polymerase subunits. The primers are known nucleotide sequences of the RNA polymerase subunit gene (rpo A: GenBank / EMBL / DDBJ Accession J01685, rpo B, rpo C: GenBank / EMBL / DDBJ Accession J01678, rpo D: GenBank / EMBL / DDBJ Accession J01687 ).

The primers shown in SEQ ID NOS: 1 and 2 were used as primers for amplifying the rpoA gene, the primers shown in SEQ ID NOS: 3 and 4 were used as primers for amplifying the rpo BC operon, and the primers used for amplifying the rpo D gene were used. The primers shown in SEQ ID NOS: 5 and 6 were used as primers.

In order to obtain the rpoD gene, the chromosomal DNA was transformed into type III, the fragment obtained by PCR using the primers shown in SEQ ID NOs: 5 and 6 was digested with EcoRI, and the plasmid pVC7 (described later) was digested with EcoRI. Plasmid pVCD was prepared by insertion into the site. Next, in order to obtain the rpo BC gene, the chromosomal DNA was subjected to PCR using the primers shown in SEQ ID NOs: 3 and 4, and the obtained fragment was subjected to DNA blunting kit (Takara Shuzo (strain) )), And inserted into the smal site of pVCD to construct pVCBCD. Subsequently, in order to obtain the rpoA gene, PCR was performed using the chromosome DNA as type III and the primers shown in SEQ ID NOS: 1 and 2 and the obtained fragment was blunt-ended in the same manner as described above. After digesting pVCBCD with Sacl Then, the plasmid was blunt-ended in the same manner as described above, and ligated to the blunt-ended rpoA gene fragment to construct plasmid pVCBCAD.

The pVC7 is as follows, a vector one for Eshierihia coli ^{pHSG39 9 (Cm r;. Takeshita} , S. et al, Gene, 61, 63-74, (1987) see) Burebibaku Teriumu - easy to It was constructed by combining pAM330, which is a cloved plasmid of Tofarmentum. pAM330 was prepared from Brevibacterium lactofermentum ATCC13869 strain. pHSG399 is cleaved with Avail (manufactured by Takara Shuzo Co., Ltd.), which is a single-site cleavage enzyme, blunt-ended with T4 DNA polymerase, and then cut with Hindlll (manufactured by Takara Shuzo Co., Ltd.). It was connected to PAM330, which had been blunt-ended with 4 DNA polymerase. pVC7 is capable of autonomous replication in E. coli and Brevibacterium lactofamentum cells, and retains the phZ299-derived multiburg cloning site and lacZ '.

<2> Preparation of L-amino acid-producing bacteria and production of L-amino acid of coryneform bacterium transformed with RNA polymerase gene

(1) Introduction of pVCBCAD into L-glucaminic acid producing strain of coryneform bacterium and L-glucaminic acid production

Brevibacterium lactofermentum AJ13029 was transformed with plasmid pVCBCAD by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791). The strain carrying pVCBCAD was selected on a medium containing 5 mg / ml chloramphenicol. Using the obtained transformant AJ13029 / pVCBCAD, culture for producing L-glucamic acid was performed as follows. AJ13029 / pVCBCAD cells obtained by culturing in a CM2B plate medium containing 5 μg / ml chloramphenicol were inoculated into a seed culture medium containing the same drug concentration and having the composition shown in Table 1. The seed culture was obtained by shaking culture at 31.5 ° C for 24 hours. The main culture medium having the composition shown in Table 1 was dispensed at a rate of 300 ml each in a 500-ml glass jar fermenter, sterilized by heating, and then inoculated with 40 ml of the above seed culture. The culture was started at a culture temperature of 31.5 ° C with a stirring speed of 800 to 1300 rpm and an aeration rate of 1/2 to 1 / lvvm. The pH of the culture was maintained at 7.5 with ammonia gas. Eight hours after the start of the culture, the culture temperature was shifted to 37 ° C. As a control, a strain obtained by transforming a corynebacterium genus AJ13029 strain with pVC7 was used as described above. Table 1 Concentration

Component

Main culture glucose 5 g / dl 15 g / dl

KH ₂ P 0 ₄ 1 g / dl 0 2 g / dl Mg S 047 H ₂ 00 04 g / dl 0 15 g / dl Fe S 047 H2O 1 mg / dl 15 mg / dl Mn S 044 H2O 1 mg / dl 15 mg / dl Soybean protein hydrolyzate 2 ml / dl 5 ml / dl Pyotin 50 g / 1 200 g / dl Siamin hydrochloride 200 g / 1 300 〃g / dl

After completion of the culture, the accumulated amount of L-glucamic acid in the culture solution was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 2 shows the results.

株 Strain L-cu, glutamic acid production (g / L) Incubation time (h)

AJ13029 / pVC7 83 3 0

AJ13029 / pVCBCAD 89 23

(2) Introduction of pVCBCAD into L-lysine-producing strains of coryneform bacterium and production of L-lysine Brevipacterium lactofermentum AJ11082 / pVCBCAD by introducing pVCBCAD into AJ11082 in the same manner Was. AJ13029 / pVCBCAD cells obtained by culturing in CM2B plate medium containing 5 g / ml chloramphenicol were inoculated into an L-lysine production medium containing the same concentration of the drug and having the following composition. Sugar in the medium disappears at ° C Shake culture until spent. As a control, a strain obtained by transforming Brevibacterium lactofermentum AJ11082 with pVC7 was cultured in the same manner as described above.

[L-lysine production medium]

Dissolve the following ingredients (in 1 L) other than calcium carbonate, adjust to pH 8.0 with K0H, sterilize at 115 ° C for 15 minutes, and add 50 g of dry-heat-killed calcium carbonate.

100 g glucose

(NH ₄ ) ₂ S 0 ₄ 55 g

Piotin 500 fig

Thiamine 2000 mg

Nicotinamide 5 mg

Protein hydrolyzate (bean concentrate) 30 ml

After the completion of the culture of 50 g of calcium carbonate, the amount of L-lysine accumulated in the culture solution was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 3 shows the results.

Strain L-Lysine production (g / dl) Incubation time (h)

AJ13029 / pVC7 2 8.9 7 2

AJ13029 / pVCBCAD 3 0.1 6 0

(3) Introduction of pVCBCAD into L-lysine and L-glucamic acid producing strains of coryneform bacteria and simultaneous production of L-lysine and L-glucamic acid PVCBCAD was introduced into Brevibacterium lactofermentum AJ12993 in the same manner as described above to obtain AJ12993 / pVCBCAD. AJ13029 / pVCBCAD cells obtained by culturing in a CM2B plate medium containing 5 g / ml of chloramphenicol were inoculated into the L-lysine production medium containing the same concentration of the drug, 31.5. Cultured at ° C. After 12 hours from the start of the culture, the culture temperature was shifted to 34 ° C, and the culture was performed with shaking until the sugar in the medium was consumed. As a control, a strain obtained by transforming Corynepacterium bacterium AJ12993 with pVC7 was cultured in the same manner as described above.

After the completion of the culture, the accumulated amounts of L-lysine and L-glucamic acid in the culture solution were measured with a Biotech Analyzer AS210 manufactured by Asahi Kasei Corporation. Table 4 shows the results.

Bacterial strain L-lysine, production unit (g / dl) L-cu, production amount of glutamic acid (g / dl) Culture time

AJ12993 / pVC7 10 5 1 8 .9 6 0

AJ12993 / pVCBCAD 1 1.2 2 0 .1 4 5

INDUSTRIAL APPLICABILITY According to the present invention, the growth of microorganisms that produce a target substance and the productivity of the target substance can be improved.

Claims

The scope of the claims

1. A microorganism that has the ability to produce the target substance and has enhanced RNA polymerase activity.

2. The microorganism according to claim 1, wherein the target substance is an L-amino acid.

3. The microorganism according to claim 1, wherein the RNA polymerase activity is enhanced by increasing the copy number of each of the rpoA, rpoB, rpoC, and rpoD genes.

4. The microorganism according to claim 1, wherein the microorganism is a bacterium belonging to the genus Escherichia or a coryneform bacterium.

5. A target substance characterized by culturing the microorganism according to any one of claims 1 to 4 in a medium, producing and accumulating the target substance in the culture, and collecting the target substance from the culture. Manufacturing method.