WO2001005979A1 - Methode d'elaboration d'une substance cible par fermentation - Google Patents

Methode d'elaboration d'une substance cible par fermentation Download PDF

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Publication number
WO2001005979A1
WO2001005979A1 PCT/JP2000/004774 JP0004774W WO0105979A1 WO 2001005979 A1 WO2001005979 A1 WO 2001005979A1 JP 0004774 W JP0004774 W JP 0004774W WO 0105979 A1 WO0105979 A1 WO 0105979A1
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WO
WIPO (PCT)
Prior art keywords
target substance
microorganism
culture
rna polymerase
dna
Prior art date
Application number
PCT/JP2000/004774
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English (en)
Japanese (ja)
Inventor
Eiichiro Kimura
Hisao Ito
Osamu Kurahashi
Original Assignee
Ajinomoto Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to AU60184/00A priority Critical patent/AU6018400A/en
Publication of WO2001005979A1 publication Critical patent/WO2001005979A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine

Definitions

  • 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.
  • a target substance such as L-amino acid, an antibiotic, a vitamin, a growth factor, or a physiologically active substance.
  • 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.
  • 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.
  • the productivity of target substances has been remarkably improved by the above-mentioned microorganism breeding technology.
  • 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.
  • the present invention is as follows.
  • a microorganism capable of producing a target substance and having enhanced RNA polymerase activity (1) A microorganism capable of producing a target substance and having enhanced RNA polymerase activity.
  • microorganism (4) The microorganism according to (1), wherein the microorganism is a bacterium belonging to the genus Escherichia or a coryneform bacterium.
  • 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.
  • 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.
  • 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.
  • 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.
  • substances that are biosynthesized by microorganisms such as nucleic acids such as guanylic acid and inosinic acid, vitamins, antibiotics, growth factors, and bioactive substances.
  • 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.
  • 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.
  • Escherichia coli bacteria such as Escherichia coli
  • coryneform bacteria such as Brevibacterium lactofermenum
  • Bacillus bacteria such as Bacillus subtilis
  • Serratia bacteria such as Serratia marcescens.
  • 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.
  • 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, ⁇ ,? ', And HI, and HI, ⁇ , and ⁇ ' are encoded by rpoA, rpoB, and rpoC genes, respectively.
  • rpoA, rpoB and rpoC genes respectively.
  • Escherichia coli rpo B and rpo C form an operon.
  • there are a plurality of types of ⁇ 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.
  • RNA polymerase subunit 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.
  • RNA polymerase 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.
  • 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.
  • rp oA GenBank / EMBL / DDBJ Accession J01685
  • 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.
  • 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.
  • 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.
  • pAM330 see Japanese Patent Application Publication No. 58-67699
  • pHM1519 see Japanese Patent Application Publication No. 58-77895
  • 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.
  • PAJ611 Escherichia Cori AJ11884 (FERM BP-138)
  • 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.
  • a recombinant DNA 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.
  • a transformation method 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)).
  • 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.
  • Bacillus subtilis, actinomycetes, and yeast are also applicable.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the microorganism of the present invention may be provided with other properties that are favorable for production of the target substance.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • required substances such as vitamin B1, L-homoserine, and L-tyrosine, or an appropriate amount of yeast extract and the like.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 '.
  • 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.
  • culture for producing L-glucamic acid was performed as follows.
  • AJ13029 / pVCBCAD cells obtained by culturing in a CM2B plate medium containing 5 ⁇ g / 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.
  • a strain obtained by transforming a corynebacterium genus AJ13029 strain with pVC7 was used as described above. Table 1 Concentration
  • Protein hydrolyzate (bean concentrate) 30 ml
  • the culture temperature was shifted to 34 ° C, and the culture was performed with shaking until the sugar in the medium was consumed.
  • a strain obtained by transforming Corynepacterium bacterium AJ12993 with pVC7 was cultured in the same manner as described above.
  • the growth of microorganisms that produce a target substance and the productivity of the target substance can be improved.

Abstract

L'invention concerne une méthode d'élaboration d'une substance cible, cette méthode consistant tout d'abord à prendre un micro-organisme pour le mettre en culture dans un milieu, puis à élaborer et à accumuler ladite substance cible dans ce milieu. Cette méthode consiste ensuite à recueillir la substance cible obtenue à l'aide d'un micro-organisme capable de produire cette substance cible et présentant une activité d'ARN polymérase améliorée, ce qui permet d'augmenter la productivité de ladite substance cible.
PCT/JP2000/004774 1999-07-19 2000-07-14 Methode d'elaboration d'une substance cible par fermentation WO2001005979A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60184/00A AU6018400A (en) 1999-07-19 2000-07-14 Process for producing target substance by fermentation method

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JP11/205267 1999-07-19
JP20526799A JP2003204783A (ja) 1999-07-19 1999-07-19 発酵法による目的物質の製造法

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WO2001005979A1 true WO2001005979A1 (fr) 2001-01-25

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Publication number Priority date Publication date Assignee Title
JP4520219B2 (ja) * 2004-05-28 2010-08-04 協和発酵バイオ株式会社 ５−アミノレブリン酸の製造法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0041767A2 (fr) * 1980-06-06 1981-12-16 Biogen, Inc. Vecteurs améliorés, méthodes de construction de tels vecteurs et d'expression de gènes clonés
JPS62244382A (ja) * 1986-04-16 1987-10-24 Ajinomoto Co Inc 新規プロモーター及び該プロモーターを用いた遺伝子発現方法
WO1997008333A1 (fr) * 1995-08-30 1997-03-06 Ajinomoto Co., Inc. Procede de production d'acides amines levogyres

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0041767A2 (fr) * 1980-06-06 1981-12-16 Biogen, Inc. Vecteurs améliorés, méthodes de construction de tels vecteurs et d'expression de gènes clonés
JPS62244382A (ja) * 1986-04-16 1987-10-24 Ajinomoto Co Inc 新規プロモーター及び該プロモーターを用いた遺伝子発現方法
WO1997008333A1 (fr) * 1995-08-30 1997-03-06 Ajinomoto Co., Inc. Procede de production d'acides amines levogyres

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KATYA Z. ET AL.: "Recombinant RNA polymerase: inducible overexpression, purification and assembly of escherichia coli rpo gene products", GENE, vol. 89, 1990, pages 7 - 12, XP002932587 *

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JP2006129840A (ja) Ｌ−グルタミン酸の製造法

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