WO2008072891A1 - Micro-organisme presentant une activite enzymatique inactivee pour nrfe et procede de production de l-tryptophane au moyen de ce micro-organisme - Google Patents

Micro-organisme presentant une activite enzymatique inactivee pour nrfe et procede de production de l-tryptophane au moyen de ce micro-organisme Download PDF

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Publication number
WO2008072891A1
WO2008072891A1 PCT/KR2007/006462 KR2007006462W WO2008072891A1 WO 2008072891 A1 WO2008072891 A1 WO 2008072891A1 KR 2007006462 W KR2007006462 W KR 2007006462W WO 2008072891 A1 WO2008072891 A1 WO 2008072891A1
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WO
WIPO (PCT)
Prior art keywords
tryptophan
gene
producing
nrffi
microorganism
Prior art date
Application number
PCT/KR2007/006462
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English (en)
Inventor
Jong-Il Choi
Jae-Yeong Ju
Hyang Choi
Ji-Sun Lee
Young-Lyeol Yang
Jin-Ho Lee
Young-Hoon Park
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Cj Cheiljedang Corporation
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Application filed by Cj Cheiljedang Corporation filed Critical Cj Cheiljedang Corporation
Publication of WO2008072891A1 publication Critical patent/WO2008072891A1/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • 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
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/01—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with NAD+ or NADP+ as acceptor (1.7.1)
    • C12Y107/01004—Nitrite reductase [NAD(P)H] (1.7.1.4)

Definitions

  • the present invention relates to a microorganism producing L-tryptophan and a method for producing L-tryptophan using the same, more particularly a recombinant microorganism with improved L-tryptophan productivity by inactivating its nrffi gene encoding formate-dependent nitrite reductase originated from E. coli and a method for producing L-tryptophan using the same.
  • Background Art
  • L-tryptophan is one of essential amino acids, which has been used as a feed additive or a raw material for medicines including injections and health foods owing to its hypnotic or tranquilizing effect.
  • a method for producing L-tryptophan is exemplified by chemical synthesis, enzyme reaction and microorganism fermentation.
  • chemical synthesis high temperature and high pressure reaction is required and as a result both D type and L type are included in the reaction product, which makes the purification process difficult.
  • Enzyme reaction has problems of high price of indole and serine used as substrates and of instability of the enzyme, as shown in the patent description of Mitsui Toatsu (Korean Patent Publication No. 90-005773).
  • the main object of the method using an L-tryptophan analog resistant strain is to overcome the feed-back inhibition of tryptophan synthesis mediated by metabolites.
  • the main object of the method using a recombinant strain is to increase the productivity/yield of tryptophan by amplifying major enzymes, which was in fact quite successful.
  • the present invention provides a recombinant microorganism producing L-tryptophan, preferably a microorganism with improved L- tryptophan productivity resulted from the inactivation of nrffi gene therein.
  • the microorganism producing L-tryptophan can be any microorganism of E. coli, Corynebacterium sp., Serratia sp. or Providencia sp. as long as it can produce L-tryptophan, and preferably E. coli and more preferably E. coli CJ285 (Korean Patent Publication No. 10-2005-0059685) producing L-tryptophan having resistance against tryptophan hydroxamate, the tryptophan analog.
  • nrffi gene (NCBI gene ID: 16131900, SEQ. ID. NO: 7) of the present invention is known to be involved in the synthesis, binding and secretion of cytochrome c under anaerobic condition in E. coli.
  • the nrffi gene is also confirmed to be required for formate-dependent nitrite reduction along with nrfG gene.
  • inactivation indicates the deficiency of nrffi gene having intracellular activity or the mutation of nrffi to reduce the protein level encoded by the gene inside the cell. So, once nrffi gene is inactivated, nrffi expression is decreased.
  • the microorganism of the present invention is prepared by inactivating nrffi gene existing in chromosome of a microorganism having L-tryptophan productivity.
  • mutation is induced by using a ray such as UV or a chemical.
  • a strain having inactivated nrffi gene is selected.
  • the inactivation can be performed by DNA recombination technique.
  • the inactivation by DNA recombination technique can be achieved by inserting nucleotide sequence or the vector containing the nucleotide sequence having homology with the gene encoding nrffi into a target microorganism to induce homologous recombination.
  • the above nucleotide sequence or vector above can include a dominant selection marker.
  • the inactivated nrffi gene or its DNA fragment contains polynucleotide sequence having sequence homology with nrffi gene of a host, but this polynucleotide sequence has to have such mutation as truncation of double strand, deletion of base, substitution or insertion of base and inversion of the end so as to be incapable of expressing the protein encoded by nrffi gene.
  • the inactivated nrffi gene or its DNA fragment is introduced into a host cell by transformation, the inactivation is induced by mixing the DNA fragment with the strain culture.
  • the strain can be transformed due to naturally suitable for the insertion of the DNA, but it is preferable for the strain to be appropriate for the DNA insertion in advance (LeBlanc et al, Plasmid 28, 130-145, 1992; Pozzi et al, J. Bacteriol. 178, 6087-6090, 1996).
  • the strain is cultured in LB medium until the early logarithmic phase. The cultured strain is washed with distilled water and 10% glycerol and then concentrated to provide appropriate condition for DNA insertion.
  • nrffi gene or its DNA fragment is mutated by deletion of a part of nrffi of the genomic DNA or insertion of a foreign DNA fragment and the wild type chromosome copy of the sequence is inactivated and substituted.
  • the nrffi inactivated DNA fragment can inactivate the wild type genome sequence with the tail sequence of the genome DNA by homologous recombination. The inactivation can be confirmed by Southern blotting or more easily by PCR.
  • the present invention also provides a method for producing L-tryptophan with high yield by culturing the microorganism whose nrffi is inactivated.
  • the culture process of the microorganism can be performed in a proper medium and conditions known to those in the art.
  • This culture process can be properly regulated according to the strain selected by those in the art.
  • the culture method can be selected appropriately.
  • the culture method can be selected from the group consisting of batch, fed-batch and continuous cultures, but not always limited thereto.
  • a variety of culture methods are described in the following reference: "Biochemical Engineering” by James M. Lee, Prentice-Hall International Editions, pp 138-176.
  • the medium for culture has to meet the culture conditions for a target strain.
  • the medium includes various carbon sources, nitrogen sources and trace elements.
  • the carbon source is exemplified by carbohydrate such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose; fat such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acid such as palmitic acid, stearic acid, and linoleic acid; alcohol such as glycerol and ethanol; and organic acid such as acetic acid.
  • carbohydrate such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose
  • fat such as soybean oil, sunflower oil, castor oil and coconut oil
  • fatty acid such as palmitic acid, stearic acid, and linoleic acid
  • alcohol such as glycerol and ethanol
  • organic acid such as acetic acid.
  • One of these compounds or a mixture thereof can be used as a carbon
  • the nitrogen source is exemplified by such organic nitrogen source as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and bean flour and such inorganic nitrogen source as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • organic nitrogen source as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and bean flour
  • inorganic nitrogen source as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the medium herein can additionally include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium- containing salts as a phosphate source.
  • the medium also can include a metal salt such as magnesium sulfate or iron sulfate.
  • amino acids, vitamins and proper precursors can be added as well.
  • PH of the culture can be adjusted during the cultivation by adding such a compound as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid.
  • the generation of air bubbles can be inhibited during the cultivation by using an antifoaming agent such as fatty acid poly glycol ester.
  • oxygen or oxygen-containing gas can be injected into the culture.
  • the temperature of the culture is preferably 20 - 45 0 C, more preferably 25 - 4O 0 C.
  • the cultivation can be continued until the production of L-tryptophan reaches a wanted level, and the preferable culture time is 10 - 160 hours.
  • L-tryptophan can be easily separated from the culture by the conventional method well known to those in the art, which is exemplified by centrifugation, filtration, ion exchange chromatography and crystallization.
  • the culture proceeds to low speed centrifugation to eliminate biomass and the obtained supernatant proceeds to ion exchange chromatography for the separation.
  • Example 1 Construction of a recombinant microorganism producing L-trvptophan whose nrfE is inactivated [29] In this example, nrffi gene of E. coli was inactivated by homologous recombination.
  • the nrffi gene (NCBI gene ID: 1631900) (SEQ. ID. NO: 7) is known to be involved in the synthesis, binding and secretion of cytochrome c in anaerobic condition. It was presumably not necessary for the culture condition for producing L-tryptophan, so that this gene was selected as a target for inactivation to minimize energy loss by unnecessary protein synthesis.
  • the nrffi gene is represented by SEQ. ID. NO: 7.
  • KCCM Korean Culture Center of Microorganisms
  • KFCC Korean Federation of Culture Collection
  • International Depository Authority located at 361-221, Hongje-1-Dong, Seodaemungu-Gu, Seoul, Korea, on November 28, 2006 (Accession No: KCCM 10805P).
  • Example 2 Comparison of L-tryptophan productivity of the recombinant mi- croorganism whose nrfE is inactivated
  • the recombinant strain CO01-0013 (KCCM 10805P) prepared in example 1 was cultured in L-tryptophan flask titer medium comprising the composition shown in Table 4 at 3O 0 C for 48 hours with stirring at 200 rpm.
  • the con- centration of tryptophan obtained from the culture and the concentration of tryptophan obtained from the culture of E.coli CJ285 were compared.
  • the L-tryptophan concentrations were mean values of three flasks.
  • the L-tryptophan productivity can be increased by inactivating nrffi gene of the L-tryptophan producing microorganism according to the method of the present invention. More particularly, the recombinant E. coli CO01-0013 (KCCM 10805P) whose nrffi is inactivated can be prepared from E. coli CJ285 producing L-tryptophan. And the productivity of L-tryptophan can be increased by culturing the recombinant microorganism.

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

Abstract

L'invention concerne un micro-organisme produisant du L-tryptophane, ainsi qu'un procédé de production de L-tryptophane au moyen de ce micro-organisme, et plus particulièrement un micro-organisme recombiné présentant une productivité améliorée de L-tryptophane par inactivation de son gène nrffi codant une nitrite réductase sensible au formiate issue d'E. coli, ainsi qu'un procédé de production de L-tryptophane au moyen dudit micro-organisme.
PCT/KR2007/006462 2006-12-13 2007-12-12 Micro-organisme presentant une activite enzymatique inactivee pour nrfe et procede de production de l-tryptophane au moyen de ce micro-organisme WO2008072891A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020060126750A KR100850853B1 (ko) 2006-12-13 2006-12-13 ｎｒｆＥ 유전자가 불활성화된 Ｌ-트립토판 생산 미생물 및이를 이용한 Ｌ-트립토판 제조방법
KR10-2006-0126750 2006-12-13

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WO2008072891A1 true WO2008072891A1 (fr) 2008-06-19

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2801611A4 (fr) * 2012-01-06 2015-10-28 Cj Cheiljedang Corp Microorganisme apte à produire un l-acide aminé, et procédé de production d'un l-acide aminé par l'utilisation de celui-ci

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105143440B (zh) 2013-04-16 2019-06-14 Cj第一制糖株式会社 具有l-色氨酸生产力的微生物以及使用所述微生物生产l-色氨酸的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62253391A (ja) * 1986-04-24 1987-11-05 Ajinomoto Co Inc 発酵法によるｌ−トリプトフアンの製造法
US5484716A (en) * 1988-03-04 1996-01-16 Kyowa Hakko Kogyo Co., Ltd. Process for producing L-tryptophan by a Corynebacterium strain having decreased phosphoenolpyruvate carboxylase activity
US5756345A (en) * 1995-09-05 1998-05-26 Degussa Aktiengesellschaft Production of tryptophan by the bacterium Escherichia coli
US20060234358A1 (en) * 2002-05-02 2006-10-19 Britta Anderlei Method for the microbial production of aromatic amino acids and other metabolites of the aromatic amino acid biosynthetic pathway

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100434108B1 (ko) * 1996-11-28 2004-09-01 씨제이 주식회사 L-트립토판의유전공학적제조방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62253391A (ja) * 1986-04-24 1987-11-05 Ajinomoto Co Inc 発酵法によるｌ−トリプトフアンの製造法
US5484716A (en) * 1988-03-04 1996-01-16 Kyowa Hakko Kogyo Co., Ltd. Process for producing L-tryptophan by a Corynebacterium strain having decreased phosphoenolpyruvate carboxylase activity
US5756345A (en) * 1995-09-05 1998-05-26 Degussa Aktiengesellschaft Production of tryptophan by the bacterium Escherichia coli
US20060234358A1 (en) * 2002-05-02 2006-10-19 Britta Anderlei Method for the microbial production of aromatic amino acids and other metabolites of the aromatic amino acid biosynthetic pathway

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2801611A4 (fr) * 2012-01-06 2015-10-28 Cj Cheiljedang Corp Microorganisme apte à produire un l-acide aminé, et procédé de production d'un l-acide aminé par l'utilisation de celui-ci
US10041099B2 (en) 2012-01-06 2018-08-07 Cj Cheiljedang Corporation L-threonine and L-tryptophan producing bacteria strain and method of making same
US10787692B2 (en) 2012-01-06 2020-09-29 Cj Cheiljedang Corporation L-threonine and L-tryptophan producing bacteria strain and method of making same

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KR20080054481A (ko) 2008-06-18

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