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WO2000009661A1 - Nouveaux micro-organismes et methode de production de l-threonine utilisant ces micro-organismes - Google Patents

Nouveaux micro-organismes et methode de production de l-threonine utilisant ces micro-organismes Download PDF

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Publication number
WO2000009661A1
WO2000009661A1 PCT/KR1998/000341 KR9800341W WO0009661A1 WO 2000009661 A1 WO2000009661 A1 WO 2000009661A1 KR 9800341 W KR9800341 W KR 9800341W WO 0009661 A1 WO0009661 A1 WO 0009661A1
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WO
WIPO (PCT)
Prior art keywords
threonine
kccm
dsm
international
culture
Prior art date
Application number
PCT/KR1998/000341
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English (en)
Inventor
In Suk Choi
Hyo Hun Yi
Jae Chun Han
Byung Rak Lim
Bun Sam Lim
Original Assignee
Daesang 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.)
Filing date
Publication date
Application filed by Daesang Corporation filed Critical Daesang Corporation
Publication of WO2000009661A1 publication Critical patent/WO2000009661A1/fr

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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

  • the present invention relates, in general, to a novel microorganism and a method for producing L-threonine using the same and, more particularly, to a Escherichia sp. which is resistant to ⁇ -methylserine and diaminosuccinic acid and susceptible to fluoropyruvate and requires L- methionine and L-isoleucine at once for growth and a method for producing a large amount of L-threonine from the bacteria even in a poor aerobic condition.
  • L-threonine an essential amino acid
  • this amino acid is used as a component for, e.g., amino acid transfusion liquid or general amino acid tablets, and as a nutrient.
  • L-threonine because it, together with L-lysine, is used as an additive of feedstuff.
  • Japanese Pat. Publication No. Heisei 5- 10076 teaches use of a recombinant DNA which contains the genetic information for asparto kinase, homoserine kinase, homoserine dehydrogenase, and threonine synthase in production of a great quantity of threonine from a L-threonine-producing Serratia sp.
  • Japanese Pat. Publication No. Heisei 1-289493 discloses that a DNA taken from a Providencia sp. resistant to methionine metabolic antagonist is genetically engineered and used to increase the productivity of L-threonine.
  • L-threonine In order to produce L-threonine, there are used a threonine metabolic antagonist- resistant Escherichia sp. which requires methionine or diaminopimelic acid for growth in Japanese Pat. Publication No. Sho. 56-10037, and a strain which can grow in a medium of L-serine and ethionine in EP 91103569.9.
  • Escherichia sp. microorganisms are usually cultured under aeration with stirring. As the preexisting Escherichia sp. Microorganisms are proliferated, they need more oxygen.
  • the dissolved oxygen in the culture is not fully replenished by current aeration techniques, so exhaustion of the dissolved oxygen occurs, causing anaerobic fermentation.
  • acetic acid is accumulated in the culture, lowering the productivity of L-threonine.
  • the novel strain of the present invention a mutant of DSM 454, can overcome the problem attributable to the physical factor upon culturing and improve the productivity of L-threonine.
  • the citric acid cycle through which microorganisms generally obtain energy from the oxidation of sugars, is less activated in the novel strain owing to the susceptibility to fluoropyruvate than in the parent strain, so less oxygen is required for growth; and thus, the reduced number of the cycle allows phosphoenolpyruvate carboxylase to supply more L-threonine precursors.
  • the novel strain of the present invention which is susceptible to fluoropyruvate and resistant to diaminosuccinic acid and ⁇ -methylserine, is mutated from Escherichia coli ATCC 21272, which requires L- methionine and L-isoleucine at once for growth.
  • Escherichia coli ATCC 21272 is first treated with chemical mutagens, NTG (N-methyl-N -nitro-N-nitroso guanidine) and DES (diethylsulfate). After the chemical treatment, replicas of the colonies thus obtained are made on a minimal agar plate and a minimal agar plate containing 40 mM of ⁇ -methylserine and, then, incubated at 37 for 2-3 days.
  • the colonies which grow on the agar plates supplemented with ⁇ -methylserine are isolated and compared in microbiological properties with the parent strain which grows in a minimal broth.
  • This mutant strain which is resistant to ⁇ -methylserine was named DSM 454.
  • the strain DSM 454 is subjected to mutation, again, after which its replicas are made on a minimal agar plate and a minimal agar plate containing 40 mM of fluoropyruvate. After incubation at 37 °C for 2-3 days, selection is made for the colonies which grow on the plate devoid of fluoropyruvate, but not on the plate supplemented with fluoropyruvate.
  • the microbiological properties of this selected strain are compared with those of its parent strain DSM 454.
  • the same mutation procedure as above is repeated for the selected strain, followed by culturing it on an agar plate which contains 2.5 g/L of diaminosuccinic acid.
  • the colonies which grow on the agar plate are isolated
  • a complete broth for the selection of the mutant strain has a composition comprising yeast extract 1%, peptone 1.0%, beef broth 0.3%, NaCl 0.5% and glucose 0.5% at pH 7.0 while a complete agar plate comprises agar 2% additionally.
  • yeast extract 1% As described above, as much as 40 mM of each of ⁇ -methylserine and fluoropyruvate and 2.5 g/L of diaminosuccinic acid are preferably used for screening the colonies which are resistant to them.
  • composition of the minimal plates on which the microbiological properties of mutant and parent strains are compared it comprises fructose 5.0%, ammonium sulfate 1.4%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, diaminopimelic acid 100 mg L, and agar 2% at pH 7.3.
  • L-methionine 200 mg/L and L- isoleucine 200 mg/L are respectively used in order to determine whether they are needed for the growth of the novel strain.
  • the novel strain DSM 9806 of the present invention was deposited in Korean Culture Center of Microorganisms on July 16, 1998 (Deposition No. KCCM-10133). As shown in Table 1, below, the novel strain DSM 9806 (KCCM-10133) requires L-methionine and L-isoleucine for growth, like the strain ATCC 21272, and resistant to ⁇ -methylserine like the parent strain DSM 454. However, the novel strain is different from the comparative strains in that it is susceptible to fluoropyruvate as well as resistant to diaminosuccinic acid.
  • novel strain DSM 9806 (KCCM-10133) and its parent strain DSM 454, it can be measured by absorbance at 610 nm, as usual. For this, after being cultured for 72 hours with agitation, each of them is 50-folds diluted.
  • the novel strain DSM 9806 and its parent strain DSM 454 show the absorbance of 0.603 and 0.907, respectively, as measured by a spectrophotometer, such as Beckman DU-70. Concerning the accumulation concentration of L-threonine, 16.32 mg/ml are measured for the novel strain DSM 9806 while 12.35 mg/ml for the parent strain DSM 454. Accordingly, the strain of the present invention produces more L-threonine than the parent strain even in a poor growth state.
  • the novel strain requires less oxygen and the fewer numbers of the citric acid cycle allow phosphoenolpyruvate carboxylate to provide a precursor for L-threonine sufficiently.
  • the resistance to diaminosuccinic acid is thought to give a contribution to the increased productivity. That is, by virtue of the resistance to diaminosuccinic acid, the novel strain of the present invention can overcome the inhibition or suppression of the enzymes involved in L-threonine biosynthesis.
  • sucrose, glucose, raw sugar catabolites, etc are available as carbon sources while ammonia gas, ammonia water, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, etc. as nitrogen sources.
  • the culture media for the fermentation may comprise other natural nutrient sources and inorganic salts.
  • the novel strain of the present invention is cultured in a fermenting bath. Culturing is carried out at about 30 °C for 3-4 days with aeration at 0.8-1.5 vvm and stirring at 500-700 rpm. With ammonia water or liquified ammonia, the pH of the culture is adjusted to 6.5-7.0. After the completion of the fermentation, the L- threonine contained in the culture media can be isolated by adsorption to ion exchange resins. The elute from the ion exchange resins is treated with ethanol to give crude L-threonine crystals.
  • DSM 9806 (KCCM-10133) and its parent strain DSM 454.
  • Pre-culture medium composition Glucose 0.5%, Yeast Extract
  • Production medium composition Glucose 10%, Corn liquid 3%,
  • Pre-Culturing The pre-culture medium was aliquoted to 18 xl85mm test tubes by 5 ml and autoclaved at 121 °C for 15 min under pressure. After being cooled, the aliquots were inoculated with the novel strain DSM 9806 and its parent strain DSM 454 by use of a sterilized metal loop. They were incubated at 30 °C for 20 hours with shaking at 120 cycles per min. Production Culturing The threonine production media were aliquoted to 500 ml Sakaguchi flasks by 70 ml and autoclaved at 121 °C for 15 min under pressure.
  • the aliquots of the autoclaved threonine were inoculated with the pre-cultures of the novel strain DSM 9806 (KCCM-10133) and its parent strain DSM 454 at an amount of 1%.
  • the strains were incubated at 30 °C for 72 hours with shaking at 120 cycles per min.
  • L-threonine was found to be accumulated at an amount of 16.32 mg/ml in the novel strain DSM 9806 (KCCM-10133) culture and 12.35 mg/ml in the parent strain DSM 454 culture.
  • Secondary Pre-culture medium composition Glucose 2%, Corn liquid 3%, Potassium dihydrogen phosphate 0.1%, Ferrous sulfate 2 ml/L, Manganese sulfate 2 ml/L, Ammonium sulfate 0.05%, Urea 0.6%, L- methionine 200 mg/L, L-isoleucine 200 mg/L, pH 7.0.
  • Production medium composition Glucose 10%, Corn liquid 3%, Potassium dihydrogen phosphate 0.1%, Ferrous sulfate 2 ml/L, Manganese sulfate 2 ml/L, Ammonium sulfate 0.5%, L-Methionine 200 mg/L, and L- Isoleucine 200 mg/L, pH 7.0.
  • Pre-Culturing Primary pre-cultures of DSM 9806 (KCCM-10133) and DSM 454 were obtained in the same manner as that of Example I. They were inoculated at 1% in 50 ml aliquots of the secondary pre-culture media in Sakaguchi flasks, which had been autoclaved at 121 °C for 15 min.
  • Incubation was carried out at 30 °C for 24 hours with shaking at 120 cycles per min, to give secondary pre-cultures.
  • Production Culturing 2L of the production media were bottled in a 5L fermentation bath and then, autoclaved at 121 °C for 15 min under pressure.
  • the secondary cultures of DSM 9806 (KCCM-10133) and DSM 454 each were inoculated at 2% and incubated at 30 °C for 72 hours with aeration at 0.8-1.5 wm and stirring at 700 rpm.
  • Sugars were added so as to maintain the sugar concentration of the media at 1-3%.
  • the media were adjusted into pH 6.5-7.0 with ammonia water.
  • L- threonine was found to be accumulated at an amount of 80.6 mg/ml in the novel strain DSM 9806 (KCCM-10133) culture and 42.6 mg/ml in the parent strain DSM 454 culture. 1 L of each of the cultures was centrifuged to harvest the bacteria which were, then, isolated through adsorption into ion-exchange resins and purified to yield L-threonine crystals at an amount of 68.51 mg per ml ofthe culture ofDSM 9806 (KCCM-10133) and 36.21 mg per ml ofthe culture of DSM 454.
  • Inocula of DSM 9806 (KCCM-10133) and DSM 454 were obtained in the same medium conditions and pre-culturing conditions as those of Example II. The production culturing procedure of Example II was repeated, except that the shaking speed was reduced to 500 rpm. After fermentation, L-threonine was found to be accumulated at an amount of 75.41 mg/ml in the novel strain DSM 9806 (KCCM-10133) culture and 37.4 mg/ml in the parent strain DSM 454 culture.
  • DSM 454 and ATCC 21272 were cultured under the same conditions as those of Example I except for using, instead of glucose, sucrose in the pre-culture and production culture media for ATCC 21272.
  • L-threonine was found to be accumulated at an amount of 12.67 mg/ml in the DSM 454 culture and 4.02 mg/ml in the ATCC 21272 culture.
  • the novel strain ofthe present invention is so specifically altered in L-threonine biosynthesis pathway that it can overcome the conventional problem upon E. coli culturing, that is, the problem that, as the culturing proceeds, accumulation of acetic acid and reduction of L-threonine productivity result from the exhaustion of the dissolved oxygen in the culture because ofthe limit of aerating agitation. Therefore, according to the present invention, a large quantity of L- threonine can be obtained even in a poor aeration state.
  • the method ofthe invention solves the culturing problems resulting from the inevitable physical factors by altering the microbiological properties.

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Abstract

L'invention concerne un mutant de Escherichia coli (KCCM-10133) résistant à la α-méthylsérine et à l'acide diaminosuccinique, sensible à la fluoropyruvate, et réclamant à la fois de la L-méthionine et de la L-isoleucine pour sa croissance, cultivé dans un milieu contenant des sucres tels que du glucose comme source de carbone, en condition aérobique, afin d'accumuler une grande quantité de L-thréonine dans la culture.
PCT/KR1998/000341 1998-08-14 1998-10-28 Nouveaux micro-organismes et methode de production de l-threonine utilisant ces micro-organismes WO2000009661A1 (fr)

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KR1998/32950 1998-08-14
KR1019980032950A KR100270509B1 (ko) 1998-08-14 1998-08-14 신규 미생물 및 이를 이용한 엘- 쓰레오닌의 제조방법

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1710317A2 (fr) 2006-07-13 2006-10-11 Degussa GmbH Procédé de préparation de L-thréonine et L-homosérine
EP1975241A1 (fr) 2007-03-29 2008-10-01 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2036979A1 (fr) 2007-09-15 2009-03-18 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2055785A1 (fr) 2007-11-02 2009-05-06 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2060636A1 (fr) 2007-11-14 2009-05-20 Evonik Degussa GmbH Procédé de fabrication d'acides aminés L en utilisant des souches de la famille des Enterobacteriaceae améliorées
DE102008002309A1 (de) 2008-06-09 2009-12-10 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
DE102008044768A1 (de) 2008-08-28 2010-03-04 Evonik Degussa Gmbh Verfahren zur Herstellung von organisch-chemischen Verbindungen unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2267145A1 (fr) 2009-06-24 2010-12-29 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP3608409A1 (fr) 2018-08-09 2020-02-12 Evonik Operations GmbH Procédé de préparation d'acides aminés l au moyen de souches améliorées de la famille des enterobacteriaceae
US11053526B2 (en) 2018-08-09 2021-07-06 Evonik Operations Gmbh Process for preparing L amino acids using improved strains of the enterobacteriaceae family
CN115521954A (zh) * 2022-10-09 2022-12-27 南京盛德生物科技研究院有限公司 一种高丝氨酸的发酵生产工艺

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100442036B1 (ko) * 1998-10-26 2004-11-06 씨제이 주식회사 인공변이 처리된 엘-라이신 생산균주의 배양방법 및 엘-라이신의 제조방법

Citations (4)

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Publication number Priority date Publication date Assignee Title
EP0301572A2 (fr) * 1987-07-31 1989-02-01 Kyowa Hakko Kogyo Co., Ltd. Procédé pour produire de la L-thréonine par fermentation
EP0445830A2 (fr) * 1990-03-09 1991-09-11 Kyowa Hakko Kogyo Co., Ltd. Procédé pour la production de L-thréonine
EP0557996A2 (fr) * 1992-02-25 1993-09-01 Kyowa Hakko Kogyo Co., Ltd. Procédé de production d'acides aminés par fermentation
US5264353A (en) * 1985-08-23 1993-11-23 Toray Industries, Inc. Process for producing L-threonine by fermentation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5264353A (en) * 1985-08-23 1993-11-23 Toray Industries, Inc. Process for producing L-threonine by fermentation
EP0301572A2 (fr) * 1987-07-31 1989-02-01 Kyowa Hakko Kogyo Co., Ltd. Procédé pour produire de la L-thréonine par fermentation
EP0445830A2 (fr) * 1990-03-09 1991-09-11 Kyowa Hakko Kogyo Co., Ltd. Procédé pour la production de L-thréonine
EP0557996A2 (fr) * 1992-02-25 1993-09-01 Kyowa Hakko Kogyo Co., Ltd. Procédé de production d'acides aminés par fermentation

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1710317A2 (fr) 2006-07-13 2006-10-11 Degussa GmbH Procédé de préparation de L-thréonine et L-homosérine
EP1975241A1 (fr) 2007-03-29 2008-10-01 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2036979A1 (fr) 2007-09-15 2009-03-18 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102007044134A1 (de) 2007-09-15 2009-03-19 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2055785A1 (fr) 2007-11-02 2009-05-06 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102007052270A1 (de) 2007-11-02 2009-05-07 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2060636A1 (fr) 2007-11-14 2009-05-20 Evonik Degussa GmbH Procédé de fabrication d'acides aminés L en utilisant des souches de la famille des Enterobacteriaceae améliorées
DE102008002309A1 (de) 2008-06-09 2009-12-10 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2133420A1 (fr) 2008-06-09 2009-12-16 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102008044768A1 (de) 2008-08-28 2010-03-04 Evonik Degussa Gmbh Verfahren zur Herstellung von organisch-chemischen Verbindungen unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2163613A2 (fr) 2008-08-28 2010-03-17 Evonik Degussa GmbH Procédé de fabrication de liaisons organo-chimiques en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2267145A1 (fr) 2009-06-24 2010-12-29 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP3608409A1 (fr) 2018-08-09 2020-02-12 Evonik Operations GmbH Procédé de préparation d'acides aminés l au moyen de souches améliorées de la famille des enterobacteriaceae
US11053526B2 (en) 2018-08-09 2021-07-06 Evonik Operations Gmbh Process for preparing L amino acids using improved strains of the enterobacteriaceae family
CN115521954A (zh) * 2022-10-09 2022-12-27 南京盛德生物科技研究院有限公司 一种高丝氨酸的发酵生产工艺
CN115521954B (zh) * 2022-10-09 2024-04-26 南京盛德生物科技研究院有限公司 一种高丝氨酸的发酵生产工艺

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KR20000013852A (ko) 2000-03-06
KR100270509B1 (ko) 2000-11-01

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