JP2003159065A - Method for producing objective substance by fermentation method - Google Patents
Method for producing objective substance by fermentation methodInfo
- Publication number
- JP2003159065A JP2003159065A JP20526699A JP20526699A JP2003159065A JP 2003159065 A JP2003159065 A JP 2003159065A JP 20526699 A JP20526699 A JP 20526699A JP 20526699 A JP20526699 A JP 20526699A JP 2003159065 A JP2003159065 A JP 2003159065A
- Authority
- JP
- Japan
- Prior art keywords
- leu
- ala
- target substance
- microorganism
- gly
- Prior art date
- Legal status (The legal status 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 status listed.)
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Classifications
-
- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- 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
-
- 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
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、微生物を利用した
目的物質の製造法に関し、詳しくは、Ｌ−アミノ酸、抗
生物質、ビタミン、成長因子、生理活性物質などの目的
物質を微生物を利用して製造する方法において、最終目
的産物である物質の生産性を改善するための手段を開示
するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a target substance using a microorganism, and more specifically, using a microorganism for a target substance such as L-amino acid, antibiotics, vitamins, growth factors and physiologically active substances. Disclosed is a method for improving the productivity of a final product, which is a product of the present invention.
【０００２】[0002]
【従来の技術】微生物を利用した物質の製造法の代表的
なものとして発酵法によるＬ−アミノ酸の製造法が知ら
れている。Ｌ−アミノ酸は、調味料や、食品として用い
られるだけでなく、医療を目的とする様々な栄養混合物
のコンポーネントとして利用される。さらに、動物用飼
料添加物として、製薬業および化学工業における試薬と
して、微生物によるＬ−リジンやＬ−ホモセリンなどの
Ｌ−アミノ酸産生のための成長因子として利用される。
発酵法によってＬ−アミノ酸を製造できる微生物として
は、コリネ型細菌、エシェリヒア属細菌、バチルス属細
菌、セラチア属細菌等が知られている。2. Description of the Related Art A method for producing L-amino acids by fermentation is known as a typical method for producing substances using microorganisms. L-amino acids are used not only as seasonings and foods, but also as components of various nutritional mixtures for medical purposes. Further, it is used as a feed additive for animals, as a reagent in the pharmaceutical industry and chemical industry, and as a growth factor for producing L-amino acids such as L-lysine and L-homoserine by microorganisms.
Coryneform bacteria, Escherichia bacteria, Bacillus bacteria, Serratia bacteria and the like are known as microorganisms capable of producing L-amino acids by the fermentation method.
【０００３】発酵法によってＬ−アミノ酸を製造するに
は、野生型微生物（野生株）を用いる方法、野生株から
誘導された栄養要求株を用いる方法、野生株から種々の
薬剤耐性変異株として誘導された代謝調節変異株を用い
る方法、栄養要求株と代謝調節変異株の両方の性質を持
った株を用いる方法等がある。In order to produce L-amino acids by fermentation, a wild-type microorganism (wild strain) is used, an auxotrophic strain derived from the wild strain is used, and various drug-resistant mutant strains are derived from the wild strain. There is a method using the above-mentioned metabolic regulation mutant strain, a method using a strain having the properties of both an auxotrophic strain and a metabolic regulation mutant strain, and the like.
【０００４】さらに近年はＬ−アミノ酸の発酵生産に、
組換えＤＮＡ技術を用いることが行われてきた。この技
術ではＬ−アミノ酸生合成系酵素をコードする遺伝子を
増強することにより宿主微生物のＬ−アミノ酸生合成系
を強化することを、その原理としている。これらの事情
については例えば「アミノ酸発酵 学会出版センター１
９８６年」に解説されている。Furthermore, in recent years, for the fermentative production of L-amino acids,
The use of recombinant DNA technology has been practiced. The principle of this technique is to strengthen the L-amino acid biosynthesis system of the host microorganism by enhancing the gene encoding the L-amino acid biosynthesis enzyme. Regarding these circumstances, for example, “Amino Acid Fermentation Society Press Center 1
986 ”.
【０００５】また、Ｌ−アミノ酸以外にも微生物を用い
た発酵法で生産されている物質は多い。例えば抗生物質
や、ビタミン等もその例である。これらの物質の発酵生
産においても、組換えＤＮＡ技術の利用は、目的物質又
はその前駆体の生合成系酵素をコードする遺伝子の増強
が主なものである。In addition to L-amino acids, many substances are produced by fermentation using microorganisms. For example, antibiotics and vitamins are examples. Also in the fermentative production of these substances, the use of recombinant DNA technology is mainly to enhance the gene encoding the biosynthetic enzyme of the target substance or its precursor.
【０００６】一方、目的物質の細胞内から細胞外への輸
送系、すなわち排出系を強化することにより、目的物質
の生産性を向上させる技術が開示されている（特開平５
−２７６９３５号）。On the other hand, a technique has been disclosed in which the productivity of the target substance is improved by strengthening the transport system of the target substance from the inside of the cell to the outside of the cell, that is, the excretion system (Japanese Patent Laid-Open No. Hei 5 (1999) -53977).
-276935).
【０００７】しかし、目的物質の生合成系の中間体又は
基質の排出量を低下させることにより、目的物質の生産
性を向上させる試みはなされていない。However, no attempt has been made to improve the productivity of the target substance by reducing the amount of the intermediate or the substrate in the biosynthesis system of the target substance discharged.
【０００８】[0008]
【発明が解決しようとする課題】本発明は、Ｌ−アミノ
酸、抗生物質、ビタミン、成長因子、生理活性物質など
の目的物質を微生物を利用して製造する方法において、
従来の方法と異なる原理によって目的物質の生産性を改
善する方法を提供することを課題とする。The present invention provides a method for producing a target substance such as an L-amino acid, an antibiotic, a vitamin, a growth factor and a physiologically active substance using a microorganism,
An object of the present invention is to provide a method for improving the productivity of a target substance by a principle different from the conventional method.
【０００９】[0009]
【課題を解決するための手段】本発明者は、上記課題を
解決するために鋭意研究を行った結果、目的物質の生合
成系の中間体又は基質の排出量が低下した変異株又は組
換え株は、目的物質の生産性が向上することを見出し、
本発明を完成するに至った。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that mutants or recombinants in which the amount of the intermediate of the biosynthesis system of the target substance or the substrate is reduced The strain found that the productivity of the target substance was improved,
The present invention has been completed.
【００１０】すなわち本発明は、
（１）微生物を培地中に培養し、該培地中に目的物質を
生成蓄積せしめ、該培養物から目的物質を採取する、微
生物を利用した目的物質の製造法において、前記微生物
は目的物質の生合成系の中間体又は基質の排出量が低下
した変異株又は組換え株であることを特徴とする方法；
（２）前記微生物は、目的物質の生合成系の中間体又は
基質の排出系が欠損又は弱化したものである（１）記載
の方法；
（３）前記目的物質がＬ−アミノ酸である（１）記載の
方法；
（４）前記目的物質がＬ−グルタミン酸であり、その生
合成系の中間体又は基質がα−ケトグルタル酸である
（３）記載の方法；
（５）前記微生物がエシェリヒア属細菌又はコリネ型細
菌である（１）記載の方法；及び
（６）前記微生物のα−ケトグルタレートパーミアーゼ
遺伝子の変異又は破壊により目的物質の生合成系の中間
体又は基質の排出系が欠損又は弱化した（４）記載の方
法；である。That is, the present invention provides (1) a method for producing a target substance using a microorganism, which comprises culturing a microorganism in a medium, producing and accumulating the target substance in the medium, and collecting the target substance from the culture. The method is characterized in that the microorganism is a mutant strain or a recombinant strain in which the amount of the intermediate or the substrate of the biosynthesis system of the target substance is reduced; (2) The microorganism is of the biosynthesis system of the target substance The method according to (1), wherein the efflux system of the intermediate or the substrate is deficient or weakened; (3) the method according to (1), wherein the target substance is an L-amino acid; (4) the target substance is L- The method according to (3), which is glutamic acid, and the intermediate or substrate of the biosynthesis system is α-ketoglutaric acid; (5) the method according to (1), wherein the microorganism is Escherichia bacterium or coryneform bacterium; and (6) α-ke of the microorganism A; glutarate permease biosynthetic intermediates or substrates for exhaust system of a target substance by mutation or disruption of the gene is deficient or weakened (4) The method according.
【００１１】[0011]
【発明の実施の形態】以下、本発明を詳細に説明する。
本発明により製造される目的物質は、微生物によって生
産され得る物質であれば特に制限されず、例えばＬ−ス
レオニン、Ｌ−リジン、Ｌ−グルタミン酸、Ｌ−ロイシ
ン、Ｌ−イソロイシン、Ｌ−バリン、Ｌ−フェニルアラ
ニン等の種々のＬ−アミノ酸が挙げられる。その他に
も、グアニル酸、イノシン酸等の核酸類、ビタミン類、
抗生物質、成長因子、生理活性物質など、微生物により
生合成される物質のうち、その生合成の中間体又は基質
の排出系が存在するものであれば、いかなるものでも良
い。また、現在微生物を利用して生産されていない物質
であっても、その生合成の中間体又は基質の排出系が存
在するものであれば本願発明が利用できることはいうま
でもない。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The target substance produced by the present invention is not particularly limited as long as it is a substance that can be produced by a microorganism, and examples thereof include L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine, L. -Various L-amino acids such as phenylalanine. In addition, nucleic acids such as guanylic acid and inosinic acid, vitamins,
Among substances that are biosynthesized by microorganisms, such as antibiotics, growth factors, and physiologically active substances, any substance may be used as long as it has an excretion system for an intermediate or substrate of the biosynthesis. Needless to say, even if the substance is not currently produced by utilizing a microorganism, the present invention can be used as long as it has a biosynthesis intermediate or a substrate excretion system.
【００１２】尚、目的物質と、中間体又は基質とは、相
対的な概念であって、目的物質であるか、中間体又は基
質であるかは、製造しようとする対象に依存する。例え
ば、アミノ酸を製造しようとする場合にはそのアミノ酸
は目的物質であるが、アミノ酸を前駆体とするペプチド
抗生物質を製造しようとする場合は、目的物質は同抗生
物質であって、アミノ酸は中間体又は基質である。The target substance and the intermediate or substrate are relative concepts, and whether the target substance is the target substance or the intermediate or substrate depends on the object to be produced. For example, when an amino acid is to be produced, the amino acid is the target substance, but when a peptide antibiotic using the amino acid as a precursor is to be produced, the target substance is the same antibiotic and the amino acid is an intermediate substance. Body or substrate.
【００１３】本発明に用いる微生物は特に制限されず、
従来発酵法による有用物質の生産に用いられている微生
物であれば使用することができる。また、従来、産業上
利用されていない微生物であっても、目的物質を生産す
る能力を有する限り、本発明を適用することができる。
本発明の微生物は、本来目的物質を生産する能力を有す
るものであってもよいし、変異法や組換えＤＮＡ技術な
どを利用した育種により目的物質を生産する能力を付与
されたものであってもよい。The microorganism used in the present invention is not particularly limited,
Any microorganism can be used as long as it is a microorganism that has been conventionally used for producing a useful substance by a fermentation method. Further, the present invention can be applied to microorganisms that have not been conventionally used industrially as long as they have the ability to produce a target substance.
The microorganism of the present invention may originally have the ability to produce a target substance, or may have been given the ability to produce a target substance by breeding using a mutation method or recombinant DNA technology. Good.
【００１４】具体的には、エシェリヒア・コリ等のエシ
ェリヒア属細菌、ブレビバクテリウム・ラクトファーメ
ンタム等のコリネ型細菌、バチルス・サブチリス等のバ
チルス属細菌、セラチア・マルセッセンス等のセラチア
属細菌等が挙げられるが、これらに制限されない。Specific examples include Escherichia bacteria such as Escherichia coli, coryneform bacteria such as Brevibacterium lactofermentum, Bacillus bacteria such as Bacillus subtilis, and Serratia bacteria such as Serratia marcescens. However, it is not limited to these.
【００１５】より具体的には以下の菌株が挙げられる。
例えば目的物質がＬ−スレオニンの場合はエシェリヒア
・コリVKPM B-3996(RIA 1867)(米国特許第5,175,107号
参照）、コリネバクテリウム・アセトアシドフィラム A
J12318（FERM BP-1172)(米国特許第5,188,949号参照）
等であり、Ｌ−リジンの場合はエシェリヒア・コリ AJ1
1442（NRRL B-12185, FERM BP-1543)(米国特許第4,346,
170号参照）、ブレビバクテリウム・ラクトファーメン
タム AJ3990（ATCC31269)(米国特許第4,066,501号参
照）等であり、Ｌ−グルタミン酸の場合はエシェリヒア
・コリ AJ12624 (FERM BP-3853)(フランス特許出願公開
第2,680,178号参照）、エシェリヒア・コリAJ13199（FE
RM P-15573)(特開平7-203980号参照)、ブレビバクテリ
ウム・ラクトファーメンタムAJ12475（FERM BP-2922)
(米国特許第5,272,067号参照）等であり、Ｌ−ロイシン
の場合はエシェリヒア・コリ AJ11478（FERM P-5274)
(特公昭 62-34397号参照）、ブレビバクテリウム・ラク
トファーメンタム AJ3718（FERM P-2516)(米国特許第3,
970,519号参照）等であり、Ｌ−イソロイシンの場合は
エシェリヒア・コリKX141（VKPM B-4781）(欧州特許出
願公開第519,113号参照）、ブレビバクテリウム・フラ
バム AJ12149（FERM BP-759)(米国特許第4,656,135号参
照）等であり、Ｌ−バリンの場合はエシェリヒア・コリ
VL1970（VKPM B-4411)）(欧州特許出願公開第519,113
号参照）、ブレビバクテリウム・ラクトファーメンタム
AJ12341（FERM BP-1763)(米国特許第5,188,948号参
照）等であり、Ｌ−フェニルアラニンの場合は、エシェ
リヒア・コリ AJ12604（FERM BP-3579)(欧州特許出願公
開第 488,424号参照）、ブレビバクテリウム・ラクトフ
ァーメンタムAJ12637（FERM BP-4160)(フランス特許出
願公開第 2,686,898号参照）等である。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 A
J12318 (FERM BP-1172) (see US Pat.No. 5,188,949)
Etc., and in the case of L-lysine, Escherichia coli AJ1
1442 (NRRL B-12185, FERM BP-1543) (U.S. Pat.No. 4,346,
No. 170), Brevibacterium lactofermentum AJ3990 (ATCC31269) (see US Pat. No. 4,066,501), etc., and in the case of L-glutamic acid, Escherichia coli AJ12624 (FERM BP-3853) (French Patent Application Publication No. 2,680,178), Escherichia coli AJ13199 (FE
RM P-15573) (see Japanese Patent Laid-Open No. 7-203980), Brevibacterium lactofermentum AJ12475 (FERM BP-2922)
(See US Pat. No. 5,272,067) and the like, and in the case of L-leucine, Escherichia coli AJ11478 (FERM P-5274)
(See Japanese Patent Publication No. 62-34397), Brevibacterium lactofermentum AJ3718 (FERM P-2516) (U.S. Pat.
970,519) and the like, and in the case of L-isoleucine, Escherichia coli KX141 (VKPM B-4781) (see European Patent Application Publication No. 519,113), Brevibacterium flavum AJ12149 (FERM BP-759) (US Patent No. 4,656,135), etc., and in the case of L-valine, Escherichia coli
VL1970 (VKPM B-4411)) (European Patent Application Publication No. 519,113)
No.), Brevibacterium lactofermentum
AJ12341 (FERM BP-1763) (see US Pat.No. 5,188,948) and the like, and in the case of L-phenylalanine, Escherichia coli AJ12604 (FERM BP-3579) (see European Patent Application Publication No. 488,424), Brevibacterium -Lact fermentum AJ12637 (FERM BP-4160) (see French Patent Application Publication No. 2,686,898) and the like.
【００１６】本発明において、目的物質の生合成系の中
間体又は基質とは、目的物質の生合成に関わる物質であ
って、排出系が存在するものであればいかなる物質であ
ってもよい。また、中間体又は基質は、前駆体など目的
物質の固有の生合成系の中間体又は基質には限られず、
例えば目的物質がＬ−アミノ酸である場合の解糖系の中
間体や基質のように、他の物質の生合成系又は代謝系の
中間体又は基質であってもよい。また、中間体又は基質
には、目的物質の生合成反応に関与するものであれば、
プロトンや電子の供与体又は受容体等の物質も含まれ
る。さらに、上記のような中間体又は基質の生合成系の
中間体又は基質も含まれる。本発明における目的物質、
その中間体又は基質、及び排出系遺伝子の例を、表１に
示す。In the present invention, the intermediate or substrate in the biosynthesis system of the target substance may be any substance as long as it is a substance involved in the biosynthesis of the target substance and has an excretion system. Further, the intermediate or substrate is not limited to the intermediate or substrate of the biosynthesis system peculiar to the target substance such as the precursor,
For example, it may be an intermediate or substrate of a biosynthetic system or a metabolic system of another substance, such as a glycolytic intermediate or substrate when the target substance is an L-amino acid. In addition, the intermediate or substrate, if it is involved in the biosynthesis reaction of the target substance,
Also included are substances such as proton or electron donors or acceptors. Furthermore, the intermediate or substrate of the biosynthesis system of the intermediate or substrate as described above is also included. The target substance in the present invention,
Table 1 shows examples of the intermediates or substrates and efflux system genes.
【００１７】[0017]
【表１】 ──────────────────────────────────── 目的物質 中間体又は基質 排出系遺伝子 ──────────────────────────────────── L-ク゛ルタミン酸 α-ケトク゛ルタル酸 α-ケトク゛ルタレートハ゜ーミアーセ゛遺伝子(kgtP) ──────────────────────────────────── L-アミノ酸1) 乳酸 乳酸パーミアーゼ遺伝子(lactP) 2) L-アミノ酸1) グリセロール ク゛リセロール促進因子遺伝子(glpF)3) ──────────────────────────────────── 1)：Ｌ−グルタミン酸、Ｌ−リジン等 2)：Dong, J.M. et al., J. Bacteriol. 175, 6671-6678 (1993) 3)：Weissenborn, D.L. et al., J. Biol. Chem., 267, 6122-6131 (1992)[Table 1] ──────────────────────────────────── Target substance Intermediate or substrate Emission system gene ─ ─────────────────────────────────── L-glutamic acid α-ketoglutaric acid α-ketoglutarate humerase gene (kgtP) ──────────────────────────────────── L-amino acid 1) Lactate Lactate permease gene (lactP ) 2) L-amino acid 1) Glycerol Glycerol promoting factor gene (glpF) 3) ──────────────────────────────── ──── 1): L-glutamic acid, L-lysine, etc. 2): Dong, JM et al., J. Bacteriol. 175, 6671-6678 (1993) 3): Weissenborn, DL et al., J. Biol Chem., 267, 6122-6131 (1992)
【００１８】また、従来排出系が知られていない物質で
あっても、排出系が存在するものであれば、本発明を適
用することができる。例えば、kgtP遺伝子に塩基配列に
ついて、既知のデータベースに対して検索を行うと、kg
tP遺伝子と相同性を有する多くの遺伝子が見出される。
これらの遺伝子は、本発明を適用することができる可能
性が高い。The present invention can be applied to substances whose discharge system is not known so far as long as the discharge system exists. For example, if you search a known database for the nucleotide sequence of the kgtP gene,
Many genes are found with homology to the tP gene.
It is highly possible that the present invention can be applied to these genes.
【００１９】本発明に用いる微生物は、上記のような目
的物質の生合成系の中間体又は基質の排出量が低下した
変異株又は組換え株である。前記排出量が低下した株と
しては、前記中間体又は基質の排出系が欠損又は弱化し
た株が挙げられる。前記排出系が欠損又は弱化した株
は、同排出系が正常に機能しないように、同排出系に関
わる１又は２以上の遺伝子を破壊し、又は変異を起こさ
せることによって、取得することができる。The microorganism used in the present invention is a mutant strain or a recombinant strain in which the amount of excreted intermediate or substrate of the biosynthesis system of the target substance as described above is reduced. Examples of the strain with reduced excretion amount include strains in which the excretion system of the intermediate or the substrate is deleted or weakened. A strain in which the efflux system is deficient or weak can be obtained by destroying or mutating one or more genes involved in the efflux system so that the efflux system does not function normally. .
【００２０】本発明に用いる変異株は、微生物の野生株
又は目的物質の生産に好ましい変異を有する変異株を変
異処理し、前記中間体又は基質の排出量が野生株に比べ
て少なく、かつ、これらの中間体又は基質の細胞内濃度
が野生株と同等又はそれ以上である株を選択することに
よって得られる。前記中間体又は基質の排出量が野生株
に比べて少ない変異株であっても、目的物質の生合成系
が完全でないものは、本発明に用いる微生物として好ま
しくない。変異処理としては、紫外線照射またはN−メ
チル−N'−ニトロ−N−ニトロソグアニジン（NTG）もし
くは亜硝酸等の通常変異処理に用いられている変異剤に
よって微生物を処理する方法が挙げられる。The mutant strain used in the present invention is obtained by subjecting a wild strain of a microorganism or a mutant strain having a preferable mutation for the production of a target substance to a mutation treatment so that the amount of the intermediate or the substrate excreted is smaller than that of the wild strain, and It can be obtained by selecting a strain in which the intracellular concentration of these intermediates or substrates is equal to or higher than that of the wild strain. Even if the mutant strain has less excretion of the intermediate or the substrate than that of the wild strain, the one in which the biosynthesis system of the target substance is not perfect is not preferable as the microorganism used in the present invention. Examples of the mutagenesis treatment include a method of treating a microorganism with ultraviolet irradiation or a mutagenesis agent which is usually used for the mutagenesis treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid.
【００２１】また、本発明に用いる組換え株は、相同組
換えによる遺伝子破壊によって創製することができる。
例えば、排出系に関与する遺伝子（排出系遺伝子）の
５’末端部及び／又は３’末端部を欠失し、正常に機能
しないように改変した排出系遺伝子を含むＤＮＡで微生
物を形質転換し、正常に機能しない排出系遺伝子と染色
体上の排出系遺伝子との間で組換えを起こさせることに
より、染色体上の排出系遺伝子を破壊することができ
る。このような相同組換えによる遺伝子破壊は既に確立
しており、直鎖ＤＮＡを用いる方法や温度感受性複製起
点を含むプラスミドを用いる方法などによっても遺伝子
破壊を行うことができる。以下に温度感受性複製起点を
含むプラスミドを用いる方法を説明する。The recombinant strain used in the present invention can be created by gene disruption by homologous recombination.
For example, a microorganism is transformed with a DNA containing an efflux system gene that has been modified so that it does not function normally by deleting the 5'-end portion and / or the 3'-end portion of a gene involved in the efflux system (efflux system gene). By causing recombination between the efflux system gene that does not function normally and the efflux system gene on the chromosome, the efflux system gene on the chromosome can be destroyed. Such gene disruption by homologous recombination has already been established, and gene disruption can also be performed by a method using a linear DNA or a method using a plasmid containing a temperature-sensitive replication origin. The method using a plasmid containing a temperature-sensitive replication origin will be described below.
【００２２】排出系遺伝子の内部を欠失し、正常に機能
しないように改変した遺伝子（欠失型遺伝子）を含むＤ
ＮＡで微生物を形質転換し、欠失型遺伝子と染色体上の
排出系遺伝子との間で組換えを起こさせることにより、
染色体上の排出系遺伝子を破壊することができる。この
ような相同組換えによる遺伝子破壊は既に確立してお
り、直鎖ＤＮＡを用いる方法や温度感受性複製起点を含
むプラスミドを用いる方法などがある。D containing a gene (deletion type gene) in which the inside of the excretion system gene is deleted and modified so as not to function normally
By transforming a microorganism with NA and causing recombination between the deletion gene and the efflux system gene on the chromosome,
Excretion system genes on the chromosome can be destroyed. Such gene disruption by homologous recombination has already been established, and there are a method using a linear DNA and a method using a plasmid containing a temperature-sensitive replication origin.
【００２３】欠失型遺伝子を、宿主染色体上の排出系遺
伝子と置換するには以下のようにすればよい。すなわ
ち、温度感受性複製起点と欠失型遺伝子とクロラムフェ
ニコール等の薬剤に耐性を示すマーカー遺伝子とを挿入
して組換えＤＮＡを調製し、この組換えＤＮＡで微生物
を形質転換し、温度感受性複製起点が機能しない温度で
形質転換株を培養し、続いてこれを薬剤を含む培地で培
養することにより、組換えＤＮＡが染色体ＤＮＡに組み
込まれた形質転換株が得られる。In order to replace the deletion type gene with the excretion system gene on the host chromosome, the following procedure may be performed. That is, a temperature-sensitive replication origin, a deletion gene and a marker gene resistant to a drug such as chloramphenicol are inserted to prepare a recombinant DNA, and a microorganism is transformed with this recombinant DNA to obtain a temperature-sensitive gene. By culturing the transformant strain at a temperature at which the origin of replication does not function, and then culturing this in a medium containing a drug, a transformant strain in which the recombinant DNA is integrated into the chromosomal DNA can be obtained.
【００２４】こうして染色体に組換えＤＮＡが組み込ま
れた株は、染色体上にもともと存在する排出系遺伝子配
列との組換えを起こし、染色体上の排出系遺伝子と欠失
型遺伝子との融合遺伝子２個が組換えＤＮＡの他の部分
（ベクター部分、温度感受性複製起点及び薬剤耐性マー
カー）を挟んだ状態で染色体に挿入されている。したが
って、この状態では正常な排出系遺伝子が優性であるの
で、形質転換株は排出系が機能する。The strain in which the recombinant DNA has been integrated into the chromosome thus undergoes recombination with the efflux system gene originally existing on the chromosome, and two fusion genes of the efflux system gene on the chromosome and the deletion gene are generated. Is inserted into the chromosome with the other part of the recombinant DNA (vector part, temperature-sensitive replication origin and drug resistance marker) sandwiched. Therefore, in this state, since the normal efflux system gene is dominant, the efflux system functions in the transformant.
【００２５】次に、染色体ＤＮＡ上に欠失型遺伝子のみ
を残すために、２個の排出系遺伝子の組換えにより１コ
ピーの排出系遺伝子を、ベクター部分（温度感受性複製
起点及び薬剤耐性マーカーを含む）とともに染色体ＤＮ
Ａから脱落させる。その際、正常な排出系遺伝子が染色
体ＤＮＡ上に残され、欠失型遺伝子が切り出される場合
と、反対に欠失型遺伝子が染色体ＤＮＡ上に残され、正
常な排出系遺伝子が切り出される場合がある。いずれの
場合も、温度感受性複製起点が機能する温度で培養すれ
ば、切り出されたＤＮＡはプラスミド状で細胞内に保持
される。次に、温度感受性複製起点が機能しない温度で
培養すると、欠失型遺伝子が染色体ＤＮＡ上に残された
場合は、正常な排出系遺伝子を含むプラスミドが細胞か
ら脱落するため排出系は機能しないが、正常な排出系遺
伝子が染色体ＤＮＡ上に残された場合は排出系が機能す
る。したがって、目的物質の生合成系の中間体又は基質
を要求する変異株を用い、同変異株の要求性の相補を指
標として、目的とする遺伝子破壊株を選択することがで
きる。Next, in order to leave only the deletion type gene on the chromosomal DNA, one copy of the efflux system gene is recombined by recombination of the two efflux system genes, and the vector part (temperature sensitive replication origin and drug resistance marker is Including) with chromosome DN
Remove from A. At that time, there are cases where a normal efflux system gene is left on chromosomal DNA and a deletion type gene is excised, and conversely, a deletion type gene is left on chromosomal DNA and a normal efflux system gene is excised. is there. In either case, the excised DNA is retained inside the cell in the form of a plasmid if it is cultured at a temperature at which the temperature-sensitive replication origin functions. Then, when the deletion-type gene is left on the chromosomal DNA when cultured at a temperature at which the temperature-sensitive replication origin does not function, the efflux system does not function because the plasmid containing the normal efflux system gene falls off from the cell. When the normal efflux system gene is left on the chromosomal DNA, the efflux system functions. Therefore, a target gene-disrupted strain can be selected by using a mutant strain that requires an intermediate or substrate of the biosynthesis system of the target substance and using the complementation of the requirement of the mutant strain as an index.
【００２６】上記のようにして得られる排出系遺伝子破
壊株は、温度感受性複製起点が機能する温度（例えば低
温）で培養すれば排出系遺伝子を細胞内に保持し、温度
感受性複製起点が機能しない温度（例えば高温）で培養
すれば排出系遺伝子を欠損する。The efflux gene-disrupted strain obtained as described above retains the efflux gene in the cell when cultured at a temperature (eg, low temperature) at which the temperature-sensitive replication origin functions, and the temperature-sensitive replication origin does not function. When cultivated at a temperature (for example, high temperature), the efflux system gene is deleted.
【００２７】尚、本発明に用いる微生物を構築した後
に、ｒｅｃＡ-株にすると、低温で培養中にプラスミド
上の排出系遺伝子が染色体へ組み込まれるのを防ぎ、遺
伝子の脱落を確実にすることができる点で好ましい。When the recA - strain is constructed after the microorganism used in the present invention has been constructed, it is possible to prevent the efflux system gene on the plasmid from being integrated into the chromosome during the culture at low temperature and to ensure the gene loss. It is preferable because it is possible.
【００２８】排出系遺伝子として具体的には、α−ケト
グルタレートパーミアーゼ遺伝子が挙げられる。同遺伝
子は、エシェリヒア・コリではkgtPとして知られてお
り、その塩基配列も報告されている（例えば、Seol, W.
and Shatkin, A.J., Proc. Natl. Acad. Sci. USA, 8
8, 3802-3806 (1991)）。同遺伝子の塩基配列及びこの
塩基配列によってコードされ得るアミノ酸配列を、配列
表の配列番号１及び２に示す。同遺伝子の塩基配列は、
DDBJ/EMBL/GenBankにaccession D90886として登録され
ている塩基配列に含まれている。Specific examples of the efflux system gene include the α-ketoglutarate permease gene. This gene is known as kgtP in Escherichia coli, and its nucleotide sequence has also been reported (for example, Seol, W.
and Shatkin, AJ, Proc. Natl. Acad. Sci. USA, 8
8, 3802-3806 (1991)). The nucleotide sequence of the gene and the amino acid sequence that can be encoded by this nucleotide sequence are shown in SEQ ID NOs: 1 and 2 in the sequence listing. The base sequence of the gene is
Included in the nucleotide sequence registered as accession D90886 in DDBJ / EMBL / GenBank.
【００２９】エシェリヒア・コリのkgtP遺伝子は、例え
ば、エシェリヒア・コリの染色体ＤＮＡを鋳型とし、配
列表の配列番号３及び４に示す塩基配列を有するオリゴ
ヌクレオチドをプライマーとするポリメラーゼ・チェイ
ン・ターミネーション法（ＰＣＲ：polymerase chain r
eaction； White,T.J. et al., Trends Genet., 5,185
(1989)参照）によって取得することができる。The Escherichia coli kgtP gene is, for example, the polymerase chain termination method using the Escherichia coli chromosomal DNA as a template and the oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 3 and 4 of the Sequence Listing as primers. PCR: polymerase chain r
eaction; White, TJ et al., Trends Genet., 5,185
(1989)).
【００３０】温度感受性プラスミドとしては、エシェリ
ヒア・コリ等のエシェリヒア属細菌で機能するものとし
ては、ｐＨＳＧ４１５及びｐＨＳＧ４２２（Hashimoto-
Gotoh, T. et al, Gene, 16, 227-235 (1981)）が、ブ
レビバクテリウム・ラクトファーメンタム等のコリネ型
細菌で機能するものとしては、ｐＨＳ４、ｐＨＳ２２、
ｐＨＳ２３が挙げられる。また、ｐＨＳ４から切り出し
たコリネ型細菌由来の複製起点を含むＤＮＡ断片を、エ
シェリヒア・コリ用のベクターであるｐＨＳＧ３９８に
接続して得られたプラスミドｐＨＳＣ４も、同様に温度
感受性プラスミドとして本発明に使用することができ
る。ｐＨＳＣ４は、コリネ型細菌、及びエシェリヒア・
コリ中で自律増殖して、宿主にクロラムフェニコール耐
性を付与する。ｐＨＳＣ４を保持するエシェリヒア・コ
リＡＪ１２５７１は、１９９０年１０月１１日に通商産
業省工業技術院生命工学工業技術研究所に受託番号ＦＥ
ＲＭＰ−１１７６３として寄託され、１９９１年８月２
６日にブダペスト条約に基づく国際寄託に移管され、Ｆ
ＥＲＭ ＢＰ−３５２４の受託番号で寄託されている。As temperature-sensitive plasmids, those that function in bacteria belonging to the genus Escherichia such as Escherichia coli are pHSG415 and pHSG422 (Hashimoto-
Gotoh, T. et al, Gene, 16, 227-235 (1981)) has the following functions in coryneform bacteria such as Brevibacterium lactofermentum: pHS4, pHS22,
pHS23 is mentioned. Further, a plasmid pHSC4 obtained by ligating a DNA fragment containing a replication origin derived from a coryneform bacterium excised from pHS4 to pHSG398 which is a vector for Escherichia coli is also used as a temperature-sensitive plasmid in the present invention. be able to. pHSC4 is a coryneform bacterium, and Escherichia
It propagates autonomously in E. coli and confers chloramphenicol resistance on the host. Escherichia coli AJ12571, which holds pHSC4, was entrusted with FE by the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology on October 11, 1990.
Deposited as RMP-11763, August 2, 1991
Transferred to an international deposit under the Budapest Treaty on the 6th, F
It has been deposited under the deposit number of ERM BP-3524.
【００３１】これらの温度感受性プラスミドはコリネ型
細菌細胞中において、約１０〜３２℃では自律増殖でき
るが、約３４℃以上では自律増殖できない。温度感受性
複製起点を有するＤＮＡ断片は、例えば上記ｐＨＳＣ４
をＢａｍＨＩとＫｐｎＩで切り出すことによって得られ
る。These temperature-sensitive plasmids can autonomously grow in coryneform bacterial cells at about 10 to 32 ° C, but cannot autonomously grow at about 34 ° C or higher. A DNA fragment having a temperature-sensitive replication origin is, for example, the above-mentioned pHSC4
Is obtained by cutting out with BamHI and KpnI.
【００３２】尚、上記の各々のプラスミドの構築及びそ
の温度感受性複製起点を含む領域の塩基配列は、特公平
７−１０８２２８号公報に記載されている。染色体ＤＮ
Ａの調製、遺伝子断片とプラスミドとの連結、ＰＣＲ、
プラスミドＤＮＡの調製、ＤＮＡの切断及び連結、形質
転換、プライマーとして用いるオリゴヌクレオチドの設
定等の方法は、当業者によく知られている通常の方法を
採用することができる。これらの方法は、Sambrook,
J., Fritsch, E. F., and Maniatis, T., "Molecular C
loning A Laboratory Manual, Second Edition", ColdS
pring Harbor Laboratory Press (1989)等に記載されて
いる。The construction of each of the above plasmids and the nucleotide sequence of the region containing the temperature-sensitive origin of replication are described in JP-B-7-108228. Chromosome DN
Preparation of A, ligation of gene fragment and plasmid, PCR,
As a method for preparing plasmid DNA, cutting and ligation of DNA, transformation, setting an oligonucleotide used as a primer and the like, an ordinary method well known to those skilled in the art can be adopted. These methods are based on Sambrook,
J., Fritsch, EF, and Maniatis, T., "Molecular C
loning A Laboratory Manual, Second Edition ", ColdS
It is described in pring Harbor Laboratory Press (1989) and the like.
【００３３】上記のようにして得られる排出系遺伝子が
破壊された微生物は、目的物質の生合成系の中間体又は
基質の細胞外への排出量が減少するので、目的物質の生
合成に供給される中間体又は基質の量が多くなり、その
結果、目的物質の生産量が上昇する。In the microorganism obtained in the above-described manner, in which the gene for excretion system is destroyed, the amount of the intermediate or the substrate of the biosynthesis system of the target substance released to the outside of the cell is reduced, and therefore the microorganism is supplied for biosynthesis of the target substance. The amount of the produced intermediate or substrate increases, and as a result, the production amount of the target substance increases.
【００３４】本発明の微生物は、本発明の効果が損なわ
れない限り、目的物質の生合成系の中間体又は基質の排
出量が低下していることに加えて、目的物質の生合成系
酵素が増強されているなど、他の性質が付与されていて
もよい。目的物質の生合成系酵素としては、例えば目的
物質がＬ−グルタミン酸である場合には、グルタミン酸
デヒドロゲナーゼ、グルタミンシンテターゼ、グルタミ
ン酸シンターゼ、イソクエン酸デヒドロゲナーゼ、アコ
ニット酸ヒドラターゼ、クエン酸シンターゼ、ピルビン
酸カルボキシラーゼ、ホスホエノールピルビン酸カルボ
キシラーゼ、エノラーゼ、ホスホグリセロムターゼ、ホ
スホグリセリン酸キナーゼ、グリセルアルデヒド−３−
リン酸デヒドロゲナーゼ、トリオースリン酸イソメラー
ゼ、フルクトースビスリン酸アルドラーゼ、ホスホフル
クトキナーゼ、グルコースリン酸イソメラーゼ等があ
る。As long as the effect of the present invention is not impaired, the microorganism of the present invention has reduced emission of the intermediate or substrate of the biosynthesis system of the target substance, and in addition, the enzyme of the biosynthesis system of the target substance. Other properties, such as the enhanced properties, may be imparted. Examples of the biosynthetic enzyme of the target substance include, for example, when the target substance is L-glutamic acid, glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, pyruvate carboxylase, phosphoenol. Pyruvate carboxylase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-
There are phosphate dehydrogenase, triose phosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase and the like.
【００３５】また、本発明の微生物は、目的物質の生合
成経路から分岐して目的物質以外の化合物を生成する反
応を触媒する酵素の活性が低下あるいは欠損していても
よい。例えば、目的物質がＬ−グルタミン酸である場合
には、前記酵素としては、α−ケトグルタル酸デヒドロ
ゲナーゼ、イソクエン酸リアーゼ、リン酸アセチルトラ
ンスフェラーゼ、酢酸キナーゼ、アセトヒドロキシ酸シ
ンターゼ、アセト乳酸シンターゼ、ギ酸アセチルトラン
スフェラーゼ、乳酸デヒドロゲナーゼ、Ｌ−グルタミン
酸デカルボキシラーゼ、１−ピロリンデヒドロゲナーゼ
等が挙げられる。In the microorganism of the present invention, the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of a target substance to produce a compound other than the target substance may be reduced or lacking. For example, when the target substance is L-glutamic acid, examples of the enzyme include α-ketoglutarate dehydrogenase, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, Lactate dehydrogenase, L-glutamate decarboxylase, 1-pyrroline dehydrogenase and the like can be mentioned.
【００３６】さらに、本発明の微生物は、目的物質の生
産にとって好ましい他の性質が付与されていてもよい。
例えば目的物質がＬ−グルタミン酸であり、微生物がコ
リネ型細菌である場合には、界面活性剤等のビオチン作
用抑制物質に対する温度感受性変異を付与することによ
り、過剰量のビオチンを含有する培地中にてビオチン作
用抑制物質の非存在下でＬ−グルタミン酸を生産させる
ことができる（WO96/06180号参照）。Furthermore, the microorganism of the present invention may be provided with other properties that are favorable for the production of the target substance.
For example, when the target substance is L-glutamic acid and the microorganism is a coryneform bacterium, by imparting a temperature-sensitive mutation to a biotin action-inhibiting substance such as a surfactant, a medium containing an excessive amount of biotin is added. It is possible to produce L-glutamic acid in the absence of a biotin action-inhibiting substance (see WO96 / 06180).
【００３７】上記のようにして目的物質の生産能が向上
した微生物を培地中に培養し、該培地中に目的物質を生
成蓄積せしめ、該培養物から目的物質を採取することに
より、目的物質を製造することができる。培養に用いる
培地や培養条件は、用いる宿主に応じて適宜選択すれば
よい。By culturing a microorganism having an improved ability to produce a target substance as described above in a medium, allowing the target substance to be produced and accumulated in the medium, and collecting the target substance from the culture, the target substance is collected. It can be manufactured. The medium and culture conditions used for culture may be appropriately selected depending on the host used.
【００３８】上記のようにして製造される目的物質は、
必要に応じて、菌体抽出液又は培地からイオン交換クロ
マトグラフィー、ゲル濾過クロマトグラフィー、吸着ク
ロマトグラフィー、溶媒沈殿等、通常の目的物質の精製
法を用いて精製することができる。The target substance produced as described above is
If necessary, it can be purified from the cell extract or medium by a conventional method for purifying the target substance such as ion exchange chromatography, gel filtration chromatography, adsorption chromatography, solvent precipitation and the like.
【００３９】[0039]
【実施例】以下、本発明を実施例によりさらに具体的に
説明する。EXAMPLES The present invention will be described in more detail below with reference to examples.
【００４０】＜１＞エシェリヒア・コリＬ−グルタミン
酸生産菌のkgtP遺伝子破壊株の創製
E. coli K12株の全ゲノムＤＮＡを、斎藤、三浦の方法
（Biochem.Biophys.Acta.,72,619(1963)）により調製し
た。一方、公知のkgtP遺伝子の塩基配列に基づいて、配
列番号３及び４に示す配列を有する２種のプライマーを
作製した。これらを用いてＰＣＲ反応を行い、kgtP遺伝
子の増幅を行った。得られたＤＮＡを、ベクターpHSG39
9（宝酒造(株)製）のEcoRI部位に挿入し、プラスミドp3
99KGTPを得た。<1> Creation of kgtP gene-disrupted strain of Escherichia coli L-glutamic acid-producing strain Total genomic DNA of E. coli K12 strain was obtained by the method of Saito and Miura (Biochem. Biophys. Acta., 72,619 (1963)). Was prepared by. On the other hand, two types of primers having the sequences shown in SEQ ID NOs: 3 and 4 were prepared based on the known nucleotide sequence of the kgtP gene. A PCR reaction was carried out using these to amplify the kgtP gene. The obtained DNA is used as the vector pHSG39.
9 (manufactured by Takara Shuzo Co., Ltd.) was inserted into the EcoRI site and plasmid p3
I got 99KGTP.
【００４１】上記プラスミドp399KGTPを制限酵素SphI及
びNheIで切断し、Ｔ４ＤＮＡポリメラーゼで平滑末化し
た後、Ｔ４ＤＮＡリガーゼを用いてセルフライゲーショ
ンを行い、kgtP遺伝子の内部を欠失させ、p399ΔKGTPを
得た。次に、p399ΔKGTPの欠失型kgtP遺伝子を、エシェ
リヒア・コリで自律複製可能なプラスミドから取得した
自己複製能が温度感受性になった変異型の複製起点を持
つプラスミドpHSG415（Hashimoto-Gotoh, T. et al, Ge
ne, 16, 227-235 (1981)）に導入した。具体的には、p3
99ΔKGTPをEcoRIで消化し、得られた欠失型kgtP遺伝子
を含む断片を、プラスミドpHSG415のEsoRI部位に導入
し、プラスミドp415ΔKGTPを作製した。The above plasmid p399KGTP was cleaved with restriction enzymes SphI and NheI, blunt-ended with T4 DNA polymerase, and then self-ligated using T4 DNA ligase to delete the inside of the kgtP gene to obtain p399ΔKGTP. Next, the deletion of the p399ΔKGTP kgtP gene was obtained from a plasmid capable of autonomous replication in Escherichia coli. The plasmid pHSG415 (Hashimoto-Gotoh, T. et. al, Ge
ne, 16, 227-235 (1981)). Specifically, p3
99ΔKGTP was digested with EcoRI, and the resulting fragment containing the deleted kgtP gene was introduced into the EsoRI site of plasmid pHSG415 to prepare plasmid p415ΔKGTP.
【００４２】このプラスミドを用いて、エシェリヒア・
コリのＬ−グルタミン酸生産菌であるエシェリヒア・コ
リAJ13199株を形質転換し、染色体上のkgtP遺伝子を欠
失型に置換した。具体的には、プラスミドが導入された
AJ13199／p415ΔKGTPをＬＢ培地（バクトトリプトン10
g、バクトイーストエクストラクト5g、NaCl 5gを１Ｌの
水に含む）で２５℃にて６時間振とう培養した後、２５
μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地上に撒
き、４２℃で培養して形成したコロニーをプラスミド組
み込み株として取得した。次に、この株から４２℃でカ
ナマイシンに対して感受性になった株をレプリカ法によ
り取得した。この感受性株から染色体上のkgtP遺伝子の
塩基配列を調べ、同遺伝子が欠失型に置換されているこ
とを確認し、これをΔkgtP株と命名した。前記エシェリ
ヒア・コリAJ13199株（特開平7-203980号参照）は、Ｄ
Ｌ−アスパラギン酸βヒドロキサメート耐性株であり、
工業技術院生命工学工業技術研究所（郵便番号305-8566
日本国茨城県つくば市東一丁目１番３号）に受託番号FE
RMP-15573として寄託されている。Using this plasmid, Escherichia
Escherichia coli AJ13199 strain, which is an L-glutamic acid-producing bacterium of Escherichia coli, was transformed, and the kgtP gene on the chromosome was replaced with the deletion type. Specifically, the plasmid was introduced
AJ13199 / p415ΔKGTP was added to LB medium (Bactotryptone 10
g, Bacto yeast extract 5 g, and NaCl 5 g in 1 L of water) at 25 ° C. for 6 hours with shaking.
A colony formed by plating on LB agar medium containing μg / ml kanamycin and culturing at 42 ° C. was obtained as a plasmid-incorporated strain. Next, a strain which became sensitive to kanamycin at 42 ° C. was obtained from this strain by the replica method. From this susceptible strain, the nucleotide sequence of the kgtP gene on the chromosome was examined, and it was confirmed that the gene was replaced with the deletion type, and this was designated as the ΔkgtP strain. The Escherichia coli AJ13199 strain (see Japanese Patent Laid-Open No. 7-203980) is D
An L-aspartic acid β-hydroxamate resistant strain,
Institute of Industrial Science and Technology, Institute of Biotechnology (Zip code 305-8566
Contract number FE at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan
Deposited as RMP-15573.
【００４３】＜２＞kgtP遺伝子破壊株の培養液中のα−
ケトグルタル酸の測定
AJ13199株及びΔkgtP株を、グルタミン酸生産培地（組
成：グルコース４０．０ｇ／Ｌ、硫酸マグネシウム（別
殺菌）１．０ｇ／Ｌ、硫酸アンモニウム２０．０ｇ／
Ｌ、リン酸２水素カリウム１．０ｇ／Ｌ、硫酸第一鉄７
水和物１０．０ｍｇ／Ｌ、硫酸マンガン５水和物１０．
０ｍｇ／Ｌ、バクトイーストエキストラクト２．０ｇ／
Ｌ、チアミン塩酸塩１０．０ｍｇ／Ｌ、炭酸カルシウム
（乾熱殺菌）５０．０ｇ／Ｌ、ｐＨ７．０（ＫＣｌで調
整））で３７℃、４０時間培養し、培地中のα−ケトグ
ルタル酸の量を、旭化成（株）製バイオテックアナライ
ザーAS210により測定した。結果を表２に示す。<2> α-in the culture solution of the kgtP gene-disrupted strain
Measurement of ketoglutaric acid AJ13199 strain and ΔkgtP strain were treated with a glutamic acid production medium (composition: glucose 40.0 g / L, magnesium sulfate (separate sterilization) 1.0 g / L, ammonium sulfate 20.0 g /
L, potassium dihydrogen phosphate 1.0 g / L, ferrous sulfate 7
Hydrate 10.0 mg / L, manganese sulfate pentahydrate 10.
0 mg / L, Bacto yeast extract 2.0 g /
L, thiamine hydrochloride 10.0 mg / L, calcium carbonate (dry heat sterilization) 50.0 g / L, pH 7.0 (adjusted with KCl)) at 37 ° C. for 40 hours, and the α-ketoglutarate in the medium was incubated. The amount was measured by Asahi Kasei Biotech Analyzer AS210. The results are shown in Table 2.
【００４４】[0044]
【表２】 ───────────────────── 菌株 α−ケトグルタル酸（g/L） ───────────────────── AJ13199 ３．０ ΔkgtP ０．５ ─────────────────────[Table 2] ───────────────────── Strain α-ketoglutarate (g / L) ───────────────────── AJ13199 3.0 ΔkgtP 0.5 ─────────────────────
【００４５】＜３＞kgtP破壊株によるＬ−グルタミン酸
の生産
AJ13199株及びΔkgtP株を、グルタミン酸生産培地で３
７℃、４０時間培養し、培地中のＬ−グルタミン酸の量
を、旭化成（株）製バイオテックアナライザーAS-210に
より測定した。結果を表３に示す。<3> Production of L-glutamic acid by a kgtP-disrupted strain.
After culturing at 7 ° C. for 40 hours, the amount of L-glutamic acid in the medium was measured by Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. The results are shown in Table 3.
【００４６】[0046]
【表３】 ───────────────────── 菌株 Ｌ−グルタミン酸（g/L） ───────────────────── AJ13199 １９．８ ΔkgtP ２１．５ ─────────────────────[Table 3] ───────────────────── Strain L-glutamic acid (g / L) ───────────────────── AJ13199 19.8 ΔkgtP 21.5 ─────────────────────
【００４７】[0047]
【発明の効果】本発明により、目的物質の生合成系の中
間体又は基質の細胞中への排出量を減少させることがで
き、その結果、目的物質の生産性を向上させることがで
きる。EFFECTS OF THE INVENTION According to the present invention, the amount of an intermediate or substrate of a biosynthesis system of a target substance discharged into cells can be reduced, and as a result, the productivity of the target substance can be improved.
【００４８】[0048]
【配列表】 SEQUENCE LISTING <110> 味の素株式会社（Ajinomoto Co., Inc.） <120> 発酵法による目的物質の製造法 <130> P-6237 <141> 1999-07-19 <160> 4 <170> PatentIn Ver. 2.0[Sequence list] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Method of producing target substance by fermentation method <130> P-6237 <141> 1999-07-19 <160> 4 <170> PatentIn Ver. 2.0
【００４９】 <210> 1 <211> 1560 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (193)..(1488) <400> 1 ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaagctc ctcaataacc 60 attttcctgc taactaaatt catggttaag gttgcataat gatatgcaac aaatgtataa 120 tatttccttt acaaaaaaaa taaacaaaag cgaccgacaa aagcatcgga ttacggcagg 180 agacataatg gc atg gct gaa agt act gta acg gca gac agc aaa ctg aca 231 Met Ala Glu Ser Thr Val Thr Ala Asp Ser Lys Leu Thr 1 5 10 agt agt gat act cgt cgc cgc att tgg gcg att gtg ggg gcc tct tca 279 Ser Ser Asp Thr Arg Arg Arg Ile Trp Ala Ile Val Gly Ala Ser Ser 15 20 25 ggt aat ctg gtc gag tgg ttc gat ttc tat gtc tac tcg ttc tgt tca 327 Gly Asn Leu Val Glu Trp Phe Asp Phe Tyr Val Tyr Ser Phe Cys Ser 30 35 40 45 ctc tac ttt gcc cac atc ttc ttc cct tcc ggg aac acg acg act caa 375 Leu Tyr Phe Ala His Ile Phe Phe Pro Ser Gly Asn Thr Thr Thr Gln 50 55 60 cta cta caa aca gca ggt gtt ttt gct gcg gga ttc ctg atg cgc cca 423 Leu Leu Gln Thr Ala Gly Val Phe Ala Ala Gly Phe Leu Met Arg Pro 65 70 75 ata ggc ggt tgg cta ttt ggc cgc ata gcc gat aaa cat ggt cgc aaa 471 Ile Gly Gly Trp Leu Phe Gly Arg Ile Ala Asp Lys His Gly Arg Lys 80 85 90 aaa tcg atg ctg tta tcg gtg tgt atg atg tgt ttc gga tcg ctg gtt 519 Lys Ser Met Leu Leu Ser Val Cys Met Met Cys Phe Gly Ser Leu Val 95 100 105 atc gcc tgc ctc cca ggt tat gaa act ata ggt acg tgg gct ccg gca 567 Ile Ala Cys Leu Pro Gly Tyr Glu Thr Ile Gly Thr Trp Ala Pro Ala 110 115 120 125 tta ttg ctt ctc gct cgt tta ttt cag gga tta tct gtt ggc gga gaa 615 Leu Leu Leu Leu Ala Arg Leu Phe Gln Gly Leu Ser Val Gly Gly Glu 130 135 140 tat ggc acc agc gcc acc tat atg agt gaa gtt gcc gtt gaa ggg cgc 663 Tyr Gly Thr Ser Ala Thr Tyr Met Ser Glu Val Ala Val Glu Gly Arg 145 150 155 aaa ggt ttt tac gca tca ttt cag tat gtg acg ttg atc ggc gga caa 711 Lys Gly Phe Tyr Ala Ser Phe Gln Tyr Val Thr Leu Ile Gly Gly Gln 160 165 170 ctg cta gcc cta ctg gtt gtc gtg gtt tta caa cac acc atg gaa gac 759 Leu Leu Ala Leu Leu Val Val Val Val Leu Gln His Thr Met Glu Asp 175 180 185 gct gca ctc aga gag tgg gga tgg cgt att cct ttc gcg tta gga gct 807 Ala Ala Leu Arg Glu Trp Gly Trp Arg Ile Pro Phe Ala Leu Gly Ala 190 195 200 205 gtg tta gct gtt gtg gcg ttg tgg tta cgt cgt cag tta gat gaa act 855 Val Leu Ala Val Val Ala Leu Trp Leu Arg Arg Gln Leu Asp Glu Thr 210 215 220 tcg caa caa gaa acg cgc gct tta aaa gaa gct gga tct ctg aaa gga 903 Ser Gln Gln Glu Thr Arg Ala Leu Lys Glu Ala Gly Ser Leu Lys Gly 225 230 235 tta tgg cgc aat cgc cgt gca ttc atc atg gtt ctc ggt ttt acc gct 951 Leu Trp Arg Asn Arg Arg Ala Phe Ile Met Val Leu Gly Phe Thr Ala 240 245 250 gcg ggc tcc ctt tgt ttc tat acc ttc act act tat atg cag aag tat 999 Ala Gly Ser Leu Cys Phe Tyr Thr Phe Thr Thr Tyr Met Gln Lys Tyr 255 260 265 ctg gta aat act gcg gga atg cat gcc aac gtg gcg agt ggc att atg 1047 Leu Val Asn Thr Ala Gly Met His Ala Asn Val Ala Ser Gly Ile Met 270 275 280 285 act gcc gca ttg ttt gta ttc atg ctt att caa cca ctc att ggc gcg 1095 Thr Ala Ala Leu Phe Val Phe Met Leu Ile Gln Pro Leu Ile Gly Ala 290 295 300 ctg tcg gat aag att ggt cgc cgt acc tca atg tta tgt ttc ggt tcg 1143 Leu Ser Asp Lys Ile Gly Arg Arg Thr Ser Met Leu Cys Phe Gly Ser 305 310 315 ctg gca gcc att ttt acc gtt cct att ctc tca gca ttg caa aac gtt 1191 Leu Ala Ala Ile Phe Thr Val Pro Ile Leu Ser Ala Leu Gln Asn Val 320 325 330 tcc tcg cct tat gcc gct ttt ggt ctg gtg atg tgt gcc ctg ctg ata 1239 Ser Ser Pro Tyr Ala Ala Phe Gly Leu Val Met Cys Ala Leu Leu Ile 335 340 345 gtg agt ttt tat aca tca atc agt gga ata ctg aag gct gag atg ttc 1287 Val Ser Phe Tyr Thr Ser Ile Ser Gly Ile Leu Lys Ala Glu Met Phe 350 355 360 365 ccg gca cag gtt cgc gca tta ggc gtt ggt ctg tca tat gcg gtc gct 1335 Pro Ala Gln Val Arg Ala Leu Gly Val Gly Leu Ser Tyr Ala Val Ala 370 375 380 aat gct ata ttt ggt ggt tcg gcg gag tac gta gcg ttg tcg ctg aaa 1383 Asn Ala Ile Phe Gly Gly Ser Ala Glu Tyr Val Ala Leu Ser Leu Lys 385 390 395 tca ata gga atg gaa aca gcc ttc ttc tgg tat gtg acc ttg atg gcc 1431 Ser Ile Gly Met Glu Thr Ala Phe Phe Trp Tyr Val Thr Leu Met Ala 400 405 410 gtg gtg gcg ttt ctg gtt tct ttg atg cta cat cgc aaa ggg aag ggg 1479 Val Val Ala Phe Leu Val Ser Leu Met Leu His Arg Lys Gly Lys Gly 415 420 425 atg cgt ctt tagtgacggg tcagttgcca gacggtatag ccggtgcttg 1528 Met Arg Leu 430 caccggcgac atcccaggcc aaatccttcc ag 1560[0049] <210> 1 <211> 1560 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (193) .. (1488) <400> 1 ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaagctc ctcaataacc 60 attttcctgc taactaaatt catggttaag gttgcataat gatatgcaac aaatgtataa 120 tatttccttt acaaaaaaaa taaacaaaag cgaccgacaa aagcatcgga ttacggcagg 180 agacataatg gc atg gct gaa agt act gta acg gca gac agc aaa ctg aca 231 Met Ala Glu Ser Thr Val Thr Ala Asp Ser Lys Leu Thr 1 5 10 agt agt gat act cgt cgc cgc att tgg gcg att gtg ggg gcc tct tca 279 Ser Ser Asp Thr Arg Arg Arg Ile Trp Ala Ile Val Gly Ala Ser Ser 15 20 25 ggt aat ctg gtc gag tgg ttc gat ttc tat gtc tac tcg ttc tgt tca 327 Gly Asn Leu Val Glu Trp Phe Asp Phe Tyr Val Tyr Ser Phe Cys Ser 30 35 40 45 ctc tac ttt gcc cac atc ttc ttc cct tcc ggg aac acg acg act caa 375 Leu Tyr Phe Ala His Ile Phe Phe Pro Ser Gly Asn Thr Thr Thr Gln 50 55 60 cta cta caa aca gca ggt gtt ttt gct gcg gga ttc ctg atg cgc cca 423 Leu Leu Gln Thr Ala Gly Val Phe Ala Ala Gly Phe Leu Met Arg Pro 65 70 75 ata ggc ggt tgg cta ttt ggc cgc ata gcc gat aaa cat ggt cgc aaa 471 Ile Gly Gly Trp Leu Phe Gly Arg Ile Ala Asp Lys His Gly Arg Lys 80 85 90 aaa tcg atg ctg tta tcg gtg tgt atg atg tgt ttc gga tcg ctg gtt 519 Lys Ser Met Leu Leu Ser Val Cys Met Met Cys Phe Gly Ser Leu Val 95 100 105 atc gcc tgc ctc cca ggt tat gaa act ata ggt acg tgg gct ccg gca 567 Ile Ala Cys Leu Pro Gly Tyr Glu Thr Ile Gly Thr Trp Ala Pro Ala 110 115 120 125 tta ttg ctt ctc gct cgt tta ttt cag gga tta tct gtt ggc gga gaa 615 Leu Leu Leu Leu Ala Arg Leu Phe Gln Gly Leu Ser Val Gly Gly Glu 130 135 140 tat ggc acc agc gcc acc tat atg agt gaa gtt gcc gtt gaa ggg cgc 663 Tyr Gly Thr Ser Ala Thr Tyr Met Ser Glu Val Ala Val Glu Gly Arg 145 150 155 aaa ggt ttt tac gca tca ttt cag tat gtg acg ttg atc ggc gga caa 711 Lys Gly Phe Tyr Ala Ser Phe Gln Tyr Val Thr Leu Ile Gly Gly Gln 160 165 170 ctg cta gcc cta ctg gtt gtc gtg gtt tta caa cac acc atg gaa gac 759 Leu Leu Ala Leu Leu Val Val Val Val Leu Gln His Thr Met Glu Asp 175 180 185 gct gca ctc aga gag tgg gga tgg cgt att cct ttc gcg tta gga gct 807 Ala Ala Leu Arg Glu Trp Gly Trp Arg Ile Pro Phe Ala Leu Gly Ala 190 195 200 205 gtg tta gct gtt gtg gcg ttg tgg tta cgt cgt cag tta gat gaa act 855 Val Leu Ala Val Val Ala Leu Trp Leu Arg Arg Gln Leu Asp Glu Thr 210 215 220 tcg caa caa gaa acg cgc gct tta aaa gaa gct gga tct ctg aaa gga 903 Ser Gln Gln Glu Thr Arg Ala Leu Lys Glu Ala Gly Ser Leu Lys Gly 225 230 235 tta tgg cgc aat cgc cgt gca ttc atc atg gtt ctc ggt ttt acc gct 951 Leu Trp Arg Asn Arg Arg Ala Phe Ile Met Val Leu Gly Phe Thr Ala 240 245 250 gcg ggc tcc ctt tgt ttc tat acc ttc act act tat atg cag aag tat 999 Ala Gly Ser Leu Cys Phe Tyr Thr Phe Thr Thr Tyr Met Gln Lys Tyr 255 260 265 ctg gta aat act gcg gga atg cat gcc aac gtg gcg agt ggc att atg 1047 Leu Val Asn Thr Ala Gly Met His Ala Asn Val Ala Ser Gly Ile Met 270 275 280 285 act gcc gca ttg ttt gta ttc atg ctt att caa cca ctc att ggc gcg 1095 Thr Ala Ala Leu Phe Val Phe Met Leu Ile Gln Pro Leu Ile Gly Ala 290 295 300 ctg tcg gat aag att ggt cgc cgt acc tca atg tta tgt ttc ggt tcg 1143 Leu Ser Asp Lys Ile Gly Arg Arg Thr Ser Met Leu Cys Phe Gly Ser 305 310 315 ctg gca gcc att ttt acc gtt cct att ctc tca gca ttg caa aac gtt 1191 Leu Ala Ala Ile Phe Thr Val Pro Ile Leu Ser Ala Leu Gln Asn Val 320 325 330 tcc tcg cct tat gcc gct ttt ggt ctg gtg atg tgt gcc ctg ctg ata 1239 Ser Ser Pro Tyr Ala Ala Phe Gly Leu Val Met Cys Ala Leu Leu Ile 335 340 345 gtg agt ttt tat aca tca atc agt gga ata ctg aag gct gag atg ttc 1287 Val Ser Phe Tyr Thr Ser Ile Ser Gly Ile Leu Lys Ala Glu Met Phe 350 355 360 365 ccg gca cag gtt cgc gca tta ggc gtt ggt ctg tca tat gcg gtc gct 1335 Pro Ala Gln Val Arg Ala Leu Gly Val Gly Leu Ser Tyr Ala Val Ala 370 375 380 aat gct ata ttt ggt ggt tcg gcg gag tac gta gcg ttg tcg ctg aaa 1383 Asn Ala Ile Phe Gly Gly Ser Ala Glu Tyr Val Ala Leu Ser Leu Lys 385 390 395 tca ata gga atg gaa aca gcc ttc ttc tgg tat gtg acc ttg atg gcc 1431 Ser Ile Gly Met Glu Thr Ala Phe Phe Trp Tyr Val Thr Leu Met Ala 400 405 410 gtg gtg gcg ttt ctg gtt tct ttg atg cta cat cgc aaa ggg aag ggg 1479 Val Val Ala Phe Leu Val Ser Leu Met Leu His Arg Lys Gly Lys Gly 415 420 425 atg cgt ctt tagtgacggg tcagttgcca gacggtatag ccggtgcttg 1528 Met Arg Leu 430 caccggcgac atcccaggcc aaatccttcc ag 1560
【００５０】 <210> 2 <211> 432 <212> PRT <213> Escherichia coli <400> 2 Met Ala Glu Ser Thr Val Thr Ala Asp Ser Lys Leu Thr Ser Ser Asp 1 5 10 15 Thr Arg Arg Arg Ile Trp Ala Ile Val Gly Ala Ser Ser Gly Asn Leu 20 25 30 Val Glu Trp Phe Asp Phe Tyr Val Tyr Ser Phe Cys Ser Leu Tyr Phe 35 40 45 Ala His Ile Phe Phe Pro Ser Gly Asn Thr Thr Thr Gln Leu Leu Gln 50 55 60 Thr Ala Gly Val Phe Ala Ala Gly Phe Leu Met Arg Pro Ile Gly Gly 65 70 75 80 Trp Leu Phe Gly Arg Ile Ala Asp Lys His Gly Arg Lys Lys Ser Met 85 90 95 Leu Leu Ser Val Cys Met Met Cys Phe Gly Ser Leu Val Ile Ala Cys 100 105 110 Leu Pro Gly Tyr Glu Thr Ile Gly Thr Trp Ala Pro Ala Leu Leu Leu 115 120 125 Leu Ala Arg Leu Phe Gln Gly Leu Ser Val Gly Gly Glu Tyr Gly Thr 130 135 140 Ser Ala Thr Tyr Met Ser Glu Val Ala Val Glu Gly Arg Lys Gly Phe 145 150 155 160 Tyr Ala Ser Phe Gln Tyr Val Thr Leu Ile Gly Gly Gln Leu Leu Ala 165 170 175 Leu Leu Val Val Val Val Leu Gln His Thr Met Glu Asp Ala Ala Leu 180 185 190 Arg Glu Trp Gly Trp Arg Ile Pro Phe Ala Leu Gly Ala Val Leu Ala 195 200 205 Val Val Ala Leu Trp Leu Arg Arg Gln Leu Asp Glu Thr Ser Gln Gln 210 215 220 Glu Thr Arg Ala Leu Lys Glu Ala Gly Ser Leu Lys Gly Leu Trp Arg 225 230 235 240 Asn Arg Arg Ala Phe Ile Met Val Leu Gly Phe Thr Ala Ala Gly Ser 245 250 255 Leu Cys Phe Tyr Thr Phe Thr Thr Tyr Met Gln Lys Tyr Leu Val Asn 260 265 270 Thr Ala Gly Met His Ala Asn Val Ala Ser Gly Ile Met Thr Ala Ala 275 280 285 Leu Phe Val Phe Met Leu Ile Gln Pro Leu Ile Gly Ala Leu Ser Asp 290 295 300 Lys Ile Gly Arg Arg Thr Ser Met Leu Cys Phe Gly Ser Leu Ala Ala 305 310 315 320 Ile Phe Thr Val Pro Ile Leu Ser Ala Leu Gln Asn Val Ser Ser Pro 325 330 335 Tyr Ala Ala Phe Gly Leu Val Met Cys Ala Leu Leu Ile Val Ser Phe 340 345 350 Tyr Thr Ser Ile Ser Gly Ile Leu Lys Ala Glu Met Phe Pro Ala Gln 355 360 365 Val Arg Ala Leu Gly Val Gly Leu Ser Tyr Ala Val Ala Asn Ala Ile 370 375 380 Phe Gly Gly Ser Ala Glu Tyr Val Ala Leu Ser Leu Lys Ser Ile Gly 385 390 395 400 Met Glu Thr Ala Phe Phe Trp Tyr Val Thr Leu Met Ala Val Val Ala 405 410 415 Phe Leu Val Ser Leu Met Leu His Arg Lys Gly Lys Gly Met Arg Leu 420 425 430[0050] <210> 2 <211> 432 <212> PRT <213> Escherichia coli <400> 2 Met Ala Glu Ser Thr Val Thr Ala Asp Ser Lys Leu Thr Ser Ser Asp 1 5 10 15 Thr Arg Arg Arg Ile Trp Ala Ile Val Gly Ala Ser Ser Gly Asn Leu 20 25 30 Val Glu Trp Phe Asp Phe Tyr Val Tyr Ser Phe Cys Ser Leu Tyr Phe 35 40 45 Ala His Ile Phe Phe Pro Ser Gly Asn Thr Thr Thr Gln Leu Leu Gln 50 55 60 Thr Ala Gly Val Phe Ala Ala Gly Phe Leu Met Arg Pro Ile Gly Gly 65 70 75 80 Trp Leu Phe Gly Arg Ile Ala Asp Lys His Gly Arg Lys Lys Ser Met 85 90 95 Leu Leu Ser Val Cys Met Met Cys Phe Gly Ser Leu Val Ile Ala Cys 100 105 110 Leu Pro Gly Tyr Glu Thr Ile Gly Thr Trp Ala Pro Ala Leu Leu Leu 115 120 125 Leu Ala Arg Leu Phe Gln Gly Leu Ser Val Gly Gly Glu Tyr Gly Thr 130 135 140 Ser Ala Thr Tyr Met Ser Glu Val Ala Val Glu Gly Arg Lys Gly Phe 145 150 155 160 Tyr Ala Ser Phe Gln Tyr Val Thr Leu Ile Gly Gly Gln Leu Leu Ala 165 170 175 Leu Leu Val Val Val Val Leu Gln His Thr Met Glu Asp Ala Ala Leu 180 185 190 Arg Glu Trp Gly Trp Arg Ile Pro Phe Ala Leu Gly Ala Val Leu Ala 195 200 205 Val Val Ala Leu Trp Leu Arg Arg Gln Leu Asp Glu Thr Ser Gln Gln 210 215 220 Glu Thr Arg Ala Leu Lys Glu Ala Gly Ser Leu Lys Gly Leu Trp Arg 225 230 235 240 Asn Arg Arg Ala Phe Ile Met Val Leu Gly Phe Thr Ala Ala Gly Ser 245 250 255 Leu Cys Phe Tyr Thr Phe Thr Thr Tyr Met Gln Lys Tyr Leu Val Asn 260 265 270 Thr Ala Gly Met His Ala Asn Val Ala Ser Gly Ile Met Thr Ala Ala 275 280 285 Leu Phe Val Phe Met Leu Ile Gln Pro Leu Ile Gly Ala Leu Ser Asp 290 295 300 Lys Ile Gly Arg Arg Thr Ser Met Leu Cys Phe Gly Ser Leu Ala Ala 305 310 315 320 Ile Phe Thr Val Pro Ile Leu Ser Ala Leu Gln Asn Val Ser Ser Pro 325 330 335 Tyr Ala Ala Phe Gly Leu Val Met Cys Ala Leu Leu Ile Val Ser Phe 340 345 350 Tyr Thr Ser Ile Ser Gly Ile Leu Lys Ala Glu Met Phe Pro Ala Gln 355 360 365 Val Arg Ala Leu Gly Val Gly Leu Ser Tyr Ala Val Ala Asn Ala Ile 370 375 380 Phe Gly Gly Ser Ala Glu Tyr Val Ala Leu Ser Leu Lys Ser Ile Gly 385 390 395 400 Met Glu Thr Ala Phe Phe Trp Tyr Val Thr Leu Met Ala Val Val Ala 405 410 415 Phe Leu Val Ser Leu Met Leu His Arg Lys Gly Lys Gly Met Arg Leu 420 425 430
【００５１】 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 gcgcgaattc attttcctgc taactaaa 28[0051] <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 gcgcgaattc attttcctgc taactaaa 28
【００５２】 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 gcgcgaattc ctggaaggat ttggcctg 28[0052] <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 gcgcgaattc ctggaaggat ttggcctg 28
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【書類名】 受託番号変更届[Document name] Consignment number change notification
【整理番号】 Ｐ−６２３７ＪＨ[Reference number] P-6237JH
【提出日】 平成１１年８月２３日[Submission date] August 23, 1999
【旧寄託機関の名称】 通商産業省工業技術院生命工学
工業技術研究所[Former name of depositary institution] Ministry of International Trade and Industry, Institute of Industrial Science and Technology
【旧受託番号】 微工研菌寄第ＦＥＲＭ Ｐ−１
５５７３[Old contract number] Microtechnology Research Institute, Bacterial FERM P-1
5573
【新寄託機関の名称】 通商産業省工業技術院生命工学
工業技術研究所[Name of new depositary institution] Ministry of International Trade and Industry Industrial Technology Institute
【新受託番号】 ＦＥＲＭ ＢＰ−５８０７[New contract number] FERM BP-5807
───────────────────────────────────────────────────── フロントページの続き (72)発明者 倉橋 修 神奈川県川崎市川崎区鈴木町１−１味の素 株式会社発酵技術研究所内 Ｆターム(参考） 4B024 AA03 BA74 BA80 CA04 DA06 GA11 HA01 4B064 AE19 CA02 CA19 CC24 DA16 4B065 AA26X AA26Y AB01 AC20 BA02 CA17 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Osamu Kurahashi Ajinomoto 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Fermentation Technology Laboratory Co., Ltd. F-term (reference) 4B024 AA03 BA74 BA80 CA04 DA06 GA11 HA01 4B064 AE19 CA02 CA19 CC24 DA16 4B065 AA26X AA26Y AB01 AC20 BA02 CA17
Claims (6)
的物質を生成蓄積せしめ、該培養物から目的物質を採取
する、微生物を利用した目的物質の製造法において、前
記微生物は目的物質の生合成系の中間体又は基質の排出
量が低下した変異株又は組換え株であることを特徴とす
る方法。1. A method for producing a target substance using a microorganism, which comprises culturing a microorganism in a medium, allowing the target substance to be produced and accumulated in the medium, and collecting the target substance from the culture, wherein the microorganism is the target substance. The method is characterized in that it is a mutant strain or a recombinant strain in which the excretion amount of the intermediate or the substrate of the biosynthesis system of is reduced.
間体又は基質の排出系が欠損又は弱化したものである請
求項１記載の方法。2. The method according to claim 1, wherein the microorganism is one in which the intermediate of the biosynthesis system of the target substance or the excretion system of the substrate is deleted or weakened.
項１記載の方法。3. The method according to claim 1, wherein the target substance is an L-amino acid.
り、その生合成系の中間体又は基質がα−ケトグルタル
酸である請求項３記載の方法。4. The method according to claim 3, wherein the target substance is L-glutamic acid, and the intermediate or substrate of its biosynthesis system is α-ketoglutaric acid.
リネ型細菌である請求項１記載の方法。5. The method according to claim 1, wherein the microorganism is Escherichia bacterium or coryneform bacterium.
ミアーゼ遺伝子の変異又は破壊により目的物質の生合成
系の中間体又は基質の排出系が欠損又は弱化した請求項
４記載の方法。6. The method according to claim 4, wherein the intermediate or the substrate excretion system of the biosynthesis system of the target substance is deleted or weakened by mutation or disruption of the α-ketoglutarate permease gene of the microorganism.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20526699A JP2003159065A (en) | 1999-07-19 | 1999-07-19 | Method for producing objective substance by fermentation method |
| PCT/JP2000/004773 WO2001005959A1 (en) | 1999-07-19 | 2000-07-14 | Process for producing target substance by fermentation method |
| AU60183/00A AU6018300A (en) | 1999-07-19 | 2000-07-14 | Process for producing target substance by fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20526699A JP2003159065A (en) | 1999-07-19 | 1999-07-19 | Method for producing objective substance by fermentation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2003159065A true JP2003159065A (en) | 2003-06-03 |
Family
ID=16504144
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20526699A Pending JP2003159065A (en) | 1999-07-19 | 1999-07-19 | Method for producing objective substance by fermentation method |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP2003159065A (en) |
| AU (1) | AU6018300A (en) |
| WO (1) | WO2001005959A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008114721A1 (en) * | 2007-03-14 | 2008-09-25 | Ajinomoto Co., Inc. | Microorganism capable of producing l-glutamic acid-type amino acid, and method for production of amino acid |
| WO2010005099A1 (en) * | 2008-07-09 | 2010-01-14 | 味の素株式会社 | Method for producing aminohydroxybenzoic acid |
| EP3020807A4 (en) * | 2013-07-09 | 2017-05-24 | Ajinomoto Co., Inc. | Method for manufacturing useful substance |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE531803T1 (en) | 2003-06-05 | 2011-11-15 | Ajinomoto Kk | FERMENTATION PROCESS USING MODIFIED BACTERIA WITH AN INCREASED ABSORPTION OF BY-PRODUCTS |
| WO2008133161A1 (en) | 2007-04-17 | 2008-11-06 | Ajinomoto Co., Inc. | Method for production of acidic substance having carboxyl group |
| JP6623690B2 (en) * | 2015-10-30 | 2019-12-25 | 味の素株式会社 | Method for producing glutamic acid-based L-amino acid |
| WO2018091525A1 (en) | 2016-11-15 | 2018-05-24 | Danmarks Tekniske Universitet | Bacterial cells with improved tolerance to diacids |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2578474B2 (en) * | 1988-01-20 | 1997-02-05 | 協和醗酵工業株式会社 | Method for producing L-glutamic acid |
| ATE214426T1 (en) * | 1992-12-21 | 2002-03-15 | Purdue Research Foundation | UNLOCKING THE COMMON SYNTHETIC PATHWAY OF AROMATIC AMINO ACIDS |
| AU746542B2 (en) * | 1998-03-18 | 2002-05-02 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
-
1999
- 1999-07-19 JP JP20526699A patent/JP2003159065A/en active Pending
-
2000
- 2000-07-14 AU AU60183/00A patent/AU6018300A/en not_active Abandoned
- 2000-07-14 WO PCT/JP2000/004773 patent/WO2001005959A1/en active Application Filing
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008114721A1 (en) * | 2007-03-14 | 2008-09-25 | Ajinomoto Co., Inc. | Microorganism capable of producing l-glutamic acid-type amino acid, and method for production of amino acid |
| US8080396B2 (en) | 2007-03-14 | 2011-12-20 | Ajinomoto Co., Inc. | Microorganism producing an amino acid of the L-glutamic acid family and a method for producing the amino acid |
| WO2010005099A1 (en) * | 2008-07-09 | 2010-01-14 | 味の素株式会社 | Method for producing aminohydroxybenzoic acid |
| US8093346B2 (en) | 2008-07-09 | 2012-01-10 | Ajinomoto Co., Inc. | Method for producing an aminohydroxybenzoic acid-type compound |
| JP5445453B2 (en) * | 2008-07-09 | 2014-03-19 | 味の素株式会社 | Process for producing aminohydroxybenzoic acids |
| EP3020807A4 (en) * | 2013-07-09 | 2017-05-24 | Ajinomoto Co., Inc. | Method for manufacturing useful substance |
| US10047385B2 (en) | 2013-07-09 | 2018-08-14 | Ajinomoto Co., Inc. | Method for manufacturing useful substance |
| EP3521433A1 (en) * | 2013-07-09 | 2019-08-07 | Ajinomoto Co., Inc. | Process for producing l-glutamic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001005959A1 (en) | 2001-01-25 |
| AU6018300A (en) | 2001-02-05 |
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