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WO2002031172A2 - Nouvelle souche d'e. coli produisant la l-threonine - Google Patents

Nouvelle souche d'e. coli produisant la l-threonine Download PDF

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Publication number
WO2002031172A2
WO2002031172A2 PCT/EP2001/011823 EP0111823W WO0231172A2 WO 2002031172 A2 WO2002031172 A2 WO 2002031172A2 EP 0111823 W EP0111823 W EP 0111823W WO 0231172 A2 WO0231172 A2 WO 0231172A2
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WO
WIPO (PCT)
Prior art keywords
threonine
ahv
coli
production
resistant
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.)
Ceased
Application number
PCT/EP2001/011823
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German (de)
English (en)
Other versions
WO2002031172A3 (fr
Inventor
Yong Ii Hwang
Man Hyo Lee
Jong Soo Choi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
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.)
Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Priority to AU2002218232A priority Critical patent/AU2002218232A1/en
Publication of WO2002031172A2 publication Critical patent/WO2002031172A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002031172A3 publication Critical patent/WO2002031172A3/fr
Ceased legal-status Critical Current

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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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • L-Threonine which is used in the medical field for amino acid infusion and nutrients, is the essential amino acid for humans and animals, and one of the limiting amino acids in feedstuff for livestock. For this reason, threonine, that is useful as food and feed additives as lysine and methionine and is expected to demand over 10 thousand tons in the world market.
  • L-Threon.ine was formerly produced by separation from protein hydrolysates . This process was not suitable because complicated procedures were necessary for the isolation of L-threonine from a mixture of other amino acids. Approaches for applying chemical synthesis on a commercial scale failed due to high production costs. Thus, the most favorable process for the large-scale production of L-threonine will be a fermentation process.
  • L-threonine using wild-type and auxotrophic strains, and direct fermentation with routine carbon source using auxotrophs, regulatory mutants resistant to analogs of L-threonine, and genetically engineered strains.
  • auxotrophs a direct fermentation from sugars and other conventional carbon sources with regulatory mutant is currently being applied for industrial production.
  • Recombinant techniques have been applied in the breeding of L-threonine producers, and these strains has been used for large-scale production.
  • L-Threonine was produced by these auxotrophs by the elimination of negative feedback controls of related amino acids, methionine, lysine and isoleucine.
  • methionine methionine
  • lysine lysine
  • isoleucine the effect of L-threonine itself was not eliminated in E. coli, resulting in relatively low yields of L-threonine.
  • Debabov et al. obtained a producer with 20 g/1 by cloning a Hindlll DNA fragment from an AHV-resistant L-threonine producer onto a plasmid pBR322 by transforming a threonine minus mutant, and by culturing the transformant .
  • the yield was increased to 55 g/1 by cultivation with 14% sucrose.
  • hybrid plasmid which contained the DNA fragment of whole threonine operon, thrA, B, C from an AHV-resistant L-threonine producer, I5IM4 , and its producer was transformed by the hybrid plasmid.
  • the activity of homoserine dehydrogenase coded by thrA gene was 5-fold increased, yielding 13.4 g/1 of L-threonine. This production was 3-fold higher than the parental strain.
  • the yield was further inproved to 65 g/1 by cultivation in a jar fermenter with glucose feeding and high supply of oxygen.
  • Fig. 1 shows the biosynthetic pathway and regulatory mechanism of threonine from aspartate in E.coli
  • Threonine is mainly biosynthesized by four enzymes, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase encoded by the thr operon.
  • aspartokinases consist of three isoenzymes (AKI, AKII, AKIII), is inhibited by threonine, methionine and lysine, respectively.
  • Homoserine dehydrogenases consisting of two isoenzymes (HDI, HDII) , is inhibited by threonine and methionine, respectively.
  • HDI isoenzymes
  • thr operon is repressed by threonine and isoleucine. This phenomenon is called as divalent attenuation.
  • produced threonine a precursor for the biosynthesis of isoleucin .
  • E.coli strains for breeding a threonine producer are B (IF013168) as wild type, ATCC21148 (DAP", Met") and ATCC21151 (DAP " , lie”) .
  • Bacillus subtilis strains for plate assay are KCTC1328 (recE4, trpC2, thr5) and ATCC33712 (leuA8, arglS , thr5, recEA r ⁇ m , ⁇ m ) . 2 .
  • Intracellular genetic recombination has been used in bacteria on strain construction.
  • this approach did not always yield high enough productivity for industrial use, and, in addition, required much time to obtain the desired strains.
  • Serratia marcescens an improved strain generated by genetic engineering produced about 40 g/1 of threonine.
  • HT101 (DAP", Met-, lie") which demands lysine, methionine and isoleucine for growth, was selected.
  • HT101 produced 12.9 g/1 of threonine higher than mother strains .
  • mutants with resistance to high concentration of aspartate and homoserine were isolated from T101 cell.
  • MT201 showed Asp" plus Horn” and the pheno- type of MT101 It produced as much as 24 g/1 of threonine in flask cultivation.
  • MT201 showed 75,5 g/1 Threonine when cultured in a 5 1 jar fermenter and a carbon conversion efficiency of 31% based on the fed sugar.
  • This strain - Escherichia coli MT201 - has been deposited with Korean Collection of Type Cultures KCTC 18048P.
  • Met-, DAP lie
  • lie are auxotroph for methionine, diaminopimelate and isoleucine, resprectively
  • thr genes means the hybrid vector containing the DNA fragment of thr operon coding threonin biosynthetic enzymes resistant to AHV
  • c) means is mutation for increasing excretion of cellular threonine and decreasing threonine consumption but the detailed genetic background is not known in now
  • MT2Q1 In order to overproduce threonine as commercial level, further improvement of MT2Q1 is needed by recombmant DNA experiments, including cloning of AHV-resistant thr operon, and transformation of threonine producer with the constructed expression vectors.
  • AHV-resistant aspartokinase For cloning of DNA fragment containing whole thr operon coding AHV-resistant aspartokinase, homoserine dehydrogenase, homoserine kinase, AHV-resistant mutants were isolated from E.coli 13, wild type strain by spontaneous mutation on 1 mg/ml of AHV contained minimal medium. Production and excretion of threonine from isolated AHV-resistant mutants were tested by the plate assay with B subtilis KCTC1328 (Thr"). Three mutants RT101, 102, 103, showing resistance to 10 mg/ml of AHV and about 3 g/1 of threonine production, were isolated from repeated isolation with gradually increased AHV concentrations. And RT101 strain was used as a donor cell for chromosomal DNA preparation.
  • Chromosomal DNA which have been prepared from the isolated mutant RT101, was fully digested with Hindlll restriction enzyme and purified only 8 ⁇ 11 kb DNA fragments from electrophoresis agarose gol.
  • Hybrid plasmids which was constructed by inserting the Hindlll DNA fragment into pBR322-HindIII , were introduced into AHV-sensitive E.coli recipient. A number of AHV-resistant transformants were isolated. Finally, a thr expression vector containing 6.2 kb of total thr operon region introduced into the MT201.
  • mutants with insensitivity to high concentration threonine was isolated from repeating spontaneous mutation and one of them, that was showed the highest resistance, was named as E.coli MT301.
  • E.coli strains, MT201, MT301 and harbouring the plasmid with thr operon were tested with table scale jar fermenter and those results was summarized in Table 2. As shown m Table 2, thr operon contributes threonine production and cell growth. Cell growth and productivity was more improved by mutation for insensitivity to high concentration threonine. Another experiments for improving thr producer, enhanced anaplerotic sequence phathway, regulated excretion mutant are in progress.
  • Table 2 Profiles of -Threonine production in E.coli mutants harbouring the thr plasmid or not

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une bactérie du genre Escherichia qui permet, après fermentation, de produire la L-thréonine en quantité supérieure à 20 g/l, et qu'on peut obtenir en mettant en oeuvre les étapes consistant à conjuguer E. coli ATCC 21148 et E. coli ATCC 21151, à produire une mutation ultérieure et à opérer une sélection de souches négatives d'acide aminohydroxyvalérique (AHV).
PCT/EP2001/011823 2000-10-12 2001-10-12 Nouvelle souche d'e. coli produisant la l-threonine Ceased WO2002031172A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002218232A AU2002218232A1 (en) 2000-10-12 2001-10-12 L-threonine producing e. coli strain

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR2000/0060045 2000-10-12
KR20000060045A KR100389580B1 (ko) 2000-10-12 2000-10-12 Ｌ-트레오닌 생산 변이주 및 그를 이용한 ｌ-트레오닌생산방법

Publications (2)

Publication Number Publication Date
WO2002031172A2 true WO2002031172A2 (fr) 2002-04-18
WO2002031172A3 WO2002031172A3 (fr) 2003-05-30

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Country Status (3)

Country Link
KR (1) KR100389580B1 (fr)
AU (1) AU2002218232A1 (fr)
WO (1) WO2002031172A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009088216A1 (fr) * 2008-01-08 2009-07-16 Cj Cheiljedang Corp. Escherichia coli producteur de l-thréonine et procédé de production l-thréonine l'utilisant
WO2009088183A1 (fr) * 2008-01-04 2009-07-16 Cj Cheiljedang Corp. Escherichia coli ayant une capacité améliorée pour produire l-thréonine, et procédé de production de l-thréonine où escherichia coli est employé
CN115678818A (zh) * 2021-07-28 2023-02-03 梅花(上海)生物科技有限公司 一种重组微生物及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100427480B1 (ko) * 2001-01-16 2004-04-27 씨제이 주식회사 Ｌ-쓰레오닌의 제조방법

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6274294A (ja) * 1985-09-28 1987-04-06 Ajinomoto Co Inc Ｌ−スレオニンの精製方法
US5976843A (en) * 1992-04-22 1999-11-02 Ajinomoto Co., Inc. Bacterial strain of Escherichia coli BKIIM B-3996 as the producer of L-threonine
US5534421A (en) * 1991-05-30 1996-07-09 Ajinomoto Co., Inc. Production of isoleucine by escherichia coli having isoleucine auxotrophy and no negative feedback inhibition of isoleucine production
JP3006926B2 (ja) * 1991-09-04 2000-02-07 協和醗酵工業株式会社 発酵法によるｌ−スレオニンの製造法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009088183A1 (fr) * 2008-01-04 2009-07-16 Cj Cheiljedang Corp. Escherichia coli ayant une capacité améliorée pour produire l-thréonine, et procédé de production de l-thréonine où escherichia coli est employé
WO2009088216A1 (fr) * 2008-01-08 2009-07-16 Cj Cheiljedang Corp. Escherichia coli producteur de l-thréonine et procédé de production l-thréonine l'utilisant
US8455237B2 (en) 2008-01-08 2013-06-04 Cj Cheiljedang Corp. Escherichia coli strain with phosphoenolpyruvate carboxylase promoter replaced with cysteine synthase promoter and method of enhanced production of L-threonine using the same
US8486670B2 (en) 2008-01-08 2013-07-16 Cheiljedang Corp. Method of producing L-threonine using Escherichia coli strain with phosphoenolpyruvate carboxylase promoter replaced with cysteine synthase promoter
CN115678818A (zh) * 2021-07-28 2023-02-03 梅花(上海)生物科技有限公司 一种重组微生物及其应用

Also Published As

Publication number Publication date
KR100389580B1 (ko) 2003-06-25
AU2002218232A1 (en) 2002-04-22
KR20020029213A (ko) 2002-04-18
WO2002031172A3 (fr) 2003-05-30

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