WO2000017379A2 - Micro-organisme producteur de l-lysine et procede de production de l-lysine utilisant ledit micro-organisme - Google Patents

Micro-organisme producteur de l-lysine et procede de production de l-lysine utilisant ledit micro-organisme Download PDF

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Publication number
WO2000017379A2
WO2000017379A2 PCT/KR1999/000563 KR9900563W WO0017379A2 WO 2000017379 A2 WO2000017379 A2 WO 2000017379A2 KR 9900563 W KR9900563 W KR 9900563W WO 0017379 A2 WO0017379 A2 WO 0017379A2
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WO
WIPO (PCT)
Prior art keywords
lysine
producing
corynebacterium glutamicum
strain
microorganism
Prior art date
Application number
PCT/KR1999/000563
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English (en)
Other versions
WO2000017379A3 (fr
Inventor
Hyun Chul Shin
Sang Jo Lim
Jung Hwan Ko
Jae Heung Lee
Original Assignee
Cheil Jedang Corporation
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 Cheil Jedang Corporation filed Critical Cheil Jedang Corporation
Priority to AU57630/99A priority Critical patent/AU5763099A/en
Publication of WO2000017379A2 publication Critical patent/WO2000017379A2/fr
Publication of WO2000017379A3 publication Critical patent/WO2000017379A3/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
    • 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
    • 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
    • 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
    • C12R2001/15—Corynebacterium

Definitions

  • the present invention relates to a novel microorganism. More particularly, it relates to Corynebacterium glutamicum CJ31-0210 which is capable of producing a very high concentration of L-lysine. In addition, the present invention relates to a process for producing L-lysine using Corynebacterium glutamicum CJ31-0210.
  • L-lysine an essential amino acid
  • the worldwide consumption of L- lysine as a feed additives was about four hundred thousand tons in 1997 and it is now increasing about 8 to 10% a year.
  • a drastic increase in the demand for L-lysine is expected. Under these circumstances, there is a pressing need for the development of an improved fermentation process for producing L-lysine through the use of a microorganism.
  • the processes for producing L-lysine used to date include a direct fermentation using wild microorganisms, a metabolism method adding biosynthetic precursors such as ⁇ -aminoadipic acid and ⁇ -ketoadipic acid, a two-step method using mutants in which L-lysine is produced from diaminopimelic acid, a method using enzymes in which L-lysine is converted from DL- ⁇ -aminocaprolactam, and a direct fermentation method using mutants.
  • the direct fermentation method using mutants has been the most popular.
  • the synthesis of L-lysine through a diaminopimelic acid pathway (DAP pathway) in bacteria and Actinomyces is represented by the following scheme:
  • the enzyme aspartokinase is subject to a feedback inhibition by lysine and threonine.
  • the enzyme homoserine dehydrogenase is repressed by methionine and is subject to a feedback inhibition by threonine.
  • the preferred L-lysine-producing microorganisms in the direct fermentation method include an auxotroph lacking the ability to synthesize one or more essential growth factors, such as homoserine auxotroph and threonine and methionine auxotroph; a threonine-methionine sensitive mutant having the enzyme homoserine dehydrogenase sensitive to a feedback inhibition by threonine, which grows actively in minimal media but not in the presence of a small amount of threonine and methionine; and an L-lysine analogue (e.g., S( ⁇ -aminoethyl)-L- cysteine) resistance mutant in which a feedback inhibition by L-lysine and L- threonine does not occur and only the enzyme homoserine dehydrogenase is inhibited by threonine.
  • an auxotroph lacking the ability to synthesize one or more essential growth factors, such as homoserine auxotroph and threonine and methion
  • Korean Laid-open Patent No. 10-1999-049691 (laying-open date: July 5, 1999) describes a novel L-lysine producing microorganism, Corynebacterium glutamicum KFCC 11001, which is not auxotrophic to L-leucine and ferments acetic acid.
  • Corynebacterium glutamicum KFCC 11001 a mutated strain which is capable of expressing L-lysine in a yield considerably larger than that of the parent strain.
  • This muated strain is referred to as Corynebacterium glutamicum CJ31-0210.
  • the present invention provides a novel Corynebacterium glutamicum CJ31- 0210 producing a high concentration of L-lysine. Pursuant to the Budapest Treaty, this strain has been deposited with the Korean Culture Center of Microorganisms, Seoul, Korea, on September, 1999, as Accession No. KCCM-10170.
  • a strain of the present invention Corynebacterium glutamicum CJ31-0210, possesses properties similar to those of the parent strain Corynebacterium glutamicum KFCC 11001. These strains are suitable to produce L-lysine in that they are a S( ⁇ - aminoethyl)-L-cysteine resistant in which a feedback inhibition by L-lysine and L- threonine does not occur and only the enzyme homoserine dihydorogenase is inhibited by threonine. However, the present strain is distinguishable from the parent strain because it cannot ferment acetic acid.
  • NTG N- methyl-N'-nitro-N-nitrosoguanidine
  • the present invention provides a method for producing L- lysine which comprises culturing Corynebacterium glutamicum CJ31-0210, removing the strains from the culture and isolating L-lysine from the culture
  • the mutants were isolated by using minimal agar media consisting of 10.0 g of glucose (if necessary, 10 g of acetic acid), 2.0 g of (NH 4 ) SO 4 , 2.0 g of urea, 1.0 g of KH 2 PO 4 , 3.0 g of K 2 HPO 4 , 0.5 g of MgSO 4 - 7H 2 0, 10.0 mg of FeSO 4 - 7H 2 O, 10.0 mg ofMnSO 4 - 5H 2 0, 100 ⁇ g of biotin, 100 ⁇ g of thiamine- HCl, 0.1 g of CaCl 2 - 2H 2 O, 80 ⁇ g of Na 2 B 4 O 7 - 10H 2 O, 40 ⁇ g of(NH 4 ) 6 MoO 27 - 4H 2 0, 10 ⁇ g ofZnSO 4 - 7H 2 O, 300 ⁇ g ofCuSO - 7H 2 0, 10 ⁇ g ofMnCl 2 - 4H 2 0, 1 mg of FeCly 6H 2 0, 20
  • the isolated strains were evaluated by using flask fermentation media consisting of 100 g of corne molasses (as a reducing sugar), 4 g of yeast extract, 40 g of (NH ) 2 SO 4 , 4 g of urea, 1 g of KH 2 PO 4 , 2.5 g of NaCl, 0.5 g ofMgSO 4 - 7H 2 0, 10 mg ofFeSO 4 - 7H 2 0, 10 mg ofMnSO 4 - 5H 2 O, 100 ⁇ g of biotin, 200 ⁇ g of thiamine- HCl, 40 g of CaCO 4 , and 1 L of water (pH 7.0 after sterilization).
  • the concentration of L-lysine was measured by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the amount of a reducing sugar was measured by the Bertrand method.
  • the acetic acid-fermenting property of the parent strain KFCC 11001 was examined as follows. This strain was grown on LB media for 16 hours and was washed twice with a sterile physiological saline. The solution was appropriately diluted. The dilution was plated on minimal agar media containing glucose, as a carbon source, and on minimal agar media containing acetic acid, as a carbon source. Cultivation was allowed at 30°C for 4 days and the degree of growth was evaluated. The results, shown in Table 2 below, demonstrate that the strain KFCC 11001 is capable of fermenting acetic acid.
  • the strains unable to ferment acetic acid were isolated as follows.
  • the Corynebacterium glutamicum KFCC 11001 at its middle exponential growth phase in LB media was suspended in a citrate buffer solution, pH 5.5, to 10 7 -10 8 cell/ml. NTG was added to the suspension to 1,000 ⁇ g/ml.
  • the reaction solution was agitated at 30 °C for 5 minutes, and washed three times with a K 2 HPO 4 buffer solution.
  • the suspension was plated on minimal agar media containing glucose and incubated at 30°C for 7 days. All colonies formed were transferred to both minimal agar media containing glucose, and minimal agar media containing acetic acid, respectively. Five acetic acid-nonfermenting colonies were obtained, which grew on glucose-containing media but not on acetic acid-containing media.
  • the A-16 strain obtained from Example 2 was designated as CJ31-0210.
  • the CJ31-0210 strain and KFCC 11001 strain were reexamined in a 7 L fermenter as follows. Each strain was incubated under agitation in flask seed media for 20 hours. A 7 L fermenter was inoculated with the seed culture for the evaluation of the strains, with sugar and other ingredients being added during incubation.
  • the media composition and the incubation conditions were as follows:
  • the fermentation was allowed at 30°C, pH 7.2, 600 rpm, and lvvm (air volume/medium volume/minute).

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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 un nouveau micro-organisme, Corynebacterium glutamicum CJ31-0210. Cette souche n'est pas capable de faire fermenter l'acide acétique, et permet d'obtenir une très forte concentration de L-lysine. De plus, l'invention concerne un procédé de production de L-lysine, qui comporte les étapes consistant à cultiver Corynebacterium glutamicum CJ31-0210, enlever les souches de la culture et isoler la L-lysine de la culture.
PCT/KR1999/000563 1998-09-22 1999-09-17 Micro-organisme producteur de l-lysine et procede de production de l-lysine utilisant ledit micro-organisme WO2000017379A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57630/99A AU5763099A (en) 1998-09-22 1999-09-17 An l-lysine-producing microorganism and a method for producing l-lysine using said microorganism

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1998/39305 1998-09-22
KR1019980039305A KR100273950B1 (ko) 1998-09-22 1998-09-22 L-라이신을 생산하는 코리네박테리움 글루타미컴 cj31-0210및 그를 이용한 l-라이신의 생산방법

Publications (2)

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WO2000017379A2 true WO2000017379A2 (fr) 2000-03-30
WO2000017379A3 WO2000017379A3 (fr) 2000-09-21

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KR (1) KR100273950B1 (fr)
AU (1) AU5763099A (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022632A2 (fr) * 2000-09-12 2002-03-21 Degussa Ag Sequences nucleotidiques codantes pour le gene pknd
US6927052B2 (en) 2000-09-12 2005-08-09 Degussa Ag Nucleotide sequences coding for the pknD gene
CN116496950A (zh) * 2022-09-27 2023-07-28 欧铭庄生物科技(天津)有限公司滨海新区分公司 一株赖氨酸生产菌株及用途，生产赖氨酸的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100614219B1 (ko) * 2004-07-05 2006-08-21 씨제이 주식회사 대두박 산분해물을 사용한 ｌ-라이신 발효

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR940001307B1 (ko) * 1991-12-27 1994-02-19 제일제당 주식회사 L-라이신을 생산하는 신규 미생물
US5302521A (en) * 1991-10-21 1994-04-12 Kyowa Hakko Kogyo Co., Ltd. Process for producing L-lysine by iodothyronine resistant strains of Mucorynebacterium glutamicum
SK278468B6 (en) * 1992-03-11 1997-06-04 Jiri Plachy Strain of microorganism corynebacterium glutamicum ccm 4265 producing l-lysine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302521A (en) * 1991-10-21 1994-04-12 Kyowa Hakko Kogyo Co., Ltd. Process for producing L-lysine by iodothyronine resistant strains of Mucorynebacterium glutamicum
KR940001307B1 (ko) * 1991-12-27 1994-02-19 제일제당 주식회사 L-라이신을 생산하는 신규 미생물
SK278468B6 (en) * 1992-03-11 1997-06-04 Jiri Plachy Strain of microorganism corynebacterium glutamicum ccm 4265 producing l-lysine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 128, no. 8, 23 February 1998, Columbus, Ohio, US; abstract no. 86400B, PLACHY J. ET AL.: 'Corynebacterium glutamicium strain producing L-lysine' page 294; & SK 278 468 B6 (APPL 719) 11 March 1992 *
DATABASE WPI Derwent Publications Ltd., London, GB; AN 1995-012600 & KR 9 401 307 B1 (CHEIL SUGAR & CO LTD) 19 February 1994 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022632A2 (fr) * 2000-09-12 2002-03-21 Degussa Ag Sequences nucleotidiques codantes pour le gene pknd
WO2002022632A3 (fr) * 2000-09-12 2002-06-13 Degussa Sequences nucleotidiques codantes pour le gene pknd
US6927052B2 (en) 2000-09-12 2005-08-09 Degussa Ag Nucleotide sequences coding for the pknD gene
US7226763B2 (en) 2000-09-12 2007-06-05 Degussa Ag Process for preparing L-amino acids with corynebacteria with enhanced nucleotide sequences coding for pknD
CN116496950A (zh) * 2022-09-27 2023-07-28 欧铭庄生物科技(天津)有限公司滨海新区分公司 一株赖氨酸生产菌株及用途，生产赖氨酸的方法
CN116496950B (zh) * 2022-09-27 2023-10-24 欧铭庄生物科技(天津)有限公司滨海新区分公司 一株赖氨酸生产菌株及用途，生产赖氨酸的方法

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KR100273950B1 (ko) 2001-01-15
AU5763099A (en) 2000-04-10
KR20000020629A (ko) 2000-04-15
WO2000017379A3 (fr) 2000-09-21

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