WO1991003560A1 - GENE L-HicDH, SEQUENCES PARTIELLES D'ADN DU GENE, VECTEURS D'EXPRESSION AVEC LE GENE OU LES SEQUENCES PARTIELLES, SOUCHES DE E. COLI POUR LA PREPARATION DE L-HicDH, L-HicDH ET PROCEDE POUR SON APPLICATION - Google Patents

GENE L-HicDH, SEQUENCES PARTIELLES D'ADN DU GENE, VECTEURS D'EXPRESSION AVEC LE GENE OU LES SEQUENCES PARTIELLES, SOUCHES DE E. COLI POUR LA PREPARATION DE L-HicDH, L-HicDH ET PROCEDE POUR SON APPLICATION Download PDF

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Publication number
WO1991003560A1
WO1991003560A1 PCT/EP1990/001530 EP9001530W WO9103560A1 WO 1991003560 A1 WO1991003560 A1 WO 1991003560A1 EP 9001530 W EP9001530 W EP 9001530W WO 9103560 A1 WO9103560 A1 WO 9103560A1
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WO
WIPO (PCT)
Prior art keywords
gene
hicdh
dna
partial sequence
gram
Prior art date
Application number
PCT/EP1990/001530
Other languages
German (de)
English (en)
Inventor
John Collins
Ronald Frank
Hans-Philip Lerch
Original Assignee
GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
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
Priority claimed from DE19893930619 external-priority patent/DE3930619A1/de
Application filed by GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) filed Critical GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Publication of WO1991003560A1 publication Critical patent/WO1991003560A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

Definitions

  • L-HicDH gene DNA partial sequences of the gene, expression vector with the gene or the partial sequences, ⁇ . -coli strains for the production of L-HicDH, L-HicDH and processes using the same
  • L-2-hydroxyisocaproic acid dehydrogenase was also from Scbou ei al. (1984) from Lactobacillus confusus DSM 20196) isolier-.
  • This NAD (H) -dependent E z m catalyzed stereospecific and reversible reduction of various aii phatic and aromatic 2-5etocarboxylic acids, the en
  • the invention relates to the L-HicDH gene, namely positions 328 to 1261 or to 1264 or to 1267 or to 1270 or to 1273 or to 1276 of the following DNA sequence:
  • the invention relates to a partial DNA sequence of the L-HicDH gene described above, namely positions 315, 316, 317 or 318 to .328 each, this partial DNA sequence at any position by 1, 2 or 3 bases can be shortened or lengthened, or 1, 2 or 3 bases of the DNA partial sequence can be replaced by a different base.
  • the DNA partial sequence mentioned can be used for translation initiation for the production of gene products in gram-positive or gram-negative microorganisms, for example in E. coli.
  • the invention relates to a partial DNA sequence of the L-HicDH gene described above, namely approximately 60 bases from the position range from 1261 to a maximum of 1321, the partial DNA sequence being shortened by 1, 2 or 3 bases at any position or can be extended or where 1, 2 or 3 bases of the DNA partial sequence can each be replaced by a different base.
  • This partial DNA sequence can be used as a transcription terminator for the production of gene products in gram-positive or gram-negative microorganisms, for example in E. coli.
  • the partial DNA sequence attached to the 3 'terminus of the gene of the desired gene product can be used, in particular as the only transcription terminator.
  • the invention comprises expression vectors for the expression of desired gene products in gram-positive or gram-negative microorganisms, for example in E. coli, these expression vectors being a partial DNA sequence described above for translation initiation and / or DNA partial sequence described above include as a transcription terminator.
  • the invention comprises expression vectors for the expression of L-HicDH in E. coli , these vectors comprising the L-HicDH gene, which is described above.
  • the invention comprises E. coli strains for the production of L-HicDH with the aid of an expression vector comprising the L-HicDH gene described above.
  • E. coli strain DSM 5520 which has been deposited under this name with the German Collection of Microorganisms.
  • L-HicDH is produced in E. coli undegraded in the cytoplasm, yields of, for example, 35 or even more percent of the total protein being able to be achieved.
  • the invention relates to the enzyme L-HicDH of the following amino acid sequence:
  • ESGFHGVLVVISNPVDVITA 661 GAATCTGGTTTCCACGGCGTATTGGTCGTGAT ⁇ CAAACCCGGTCGACGTGATTACGGCC - -
  • PUTATIVE TERMINATOR 1261 AAACACAAAAAGTGGC ⁇ CAATCTGGTGA ⁇ GGACCACTTTTTTCTAATTGTGCATGAAA where the following letters mean:
  • Y tyrosine wherein the enzyme can be shortened or extended by 1, 2 or 3 amino acids in any position or where an amino acid can be replaced by another amino acid.
  • the invention relates to a process for converting hydroxy acids into keto acids or of keto acids into hydroxy acids, for example L-hydroxy acids, using L-HicDH, in particular if the enzyme is obtained with the aid of an E. coli strain according to the invention has been.
  • the E. coli strain 5520 according to the invention can serve as starting material in order to use routine methods to produce or use all other objects of the invention.
  • L-2-hydroxyisocaproic acid dehydrogenase from Lactobacillus confusus was purified and partially sequenced.
  • a 69-mer oligonucleotide probe corresponding to the 25 N-terminal amino acids was synthesized and a physical map was made of the genomic region that hybridized the most.
  • a strongly hybridizing restriction fragment was highly purified and finally cloned in pBR322 at a low frequency.
  • Subcloning downstream of a double P PH promoter in plasmid pJLA501 (Schauder et al. 1987) provided a highly inducible clone that forms up to 35% of the entire cell protein L-HicDH in soluble and fully active form in the cytoplasm.
  • the codon usage corresponded to that of other Lactobacillus genes that have been cloned.
  • a typical Shine-Dalgarno sequence is present and a loop structure at the 3 'end of the gene represents a strong terminator sequence.
  • the gene encodes a 34 kDa protein subunit.

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

Abstract

L'invention concerne le gène L-HicDH, des séquences partielles d'ADN du gène L-HicDH (en particulier pour l'application comme initiateur de translation et/ou termineur de transcription pour la préparation de produits de gènes), des vecteurs d'expression avec le gène L-HicDH ou les séquences partielles d'ADN, des souches de E. coli pour la préparation de L-HicDH, l'enzyme L-HicDH ainsi que des procédés de son application pour la transformation d'hydroxy-acides en céto-acides et inversement.
PCT/EP1990/001530 1989-09-11 1990-09-11 GENE L-HicDH, SEQUENCES PARTIELLES D'ADN DU GENE, VECTEURS D'EXPRESSION AVEC LE GENE OU LES SEQUENCES PARTIELLES, SOUCHES DE E. COLI POUR LA PREPARATION DE L-HicDH, L-HicDH ET PROCEDE POUR SON APPLICATION WO1991003560A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE3930317 1989-09-11
DEP3930317.9 1989-09-11
DEP3930619.4 1989-09-13
DE19893930619 DE3930619A1 (de) 1989-09-13 1989-09-13 L-hicdh-gen, dna-teilsequenzen des gens, expressionsvektoren mit dem gen oder den teilsequenzen, e.-coli-staemme fuer die herstellung von l-hicdh sowie verfahren unter dessen verwendung

Publications (1)

Publication Number Publication Date
WO1991003560A1 true WO1991003560A1 (fr) 1991-03-21

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1990/001530 WO1991003560A1 (fr) 1989-09-11 1990-09-11 GENE L-HicDH, SEQUENCES PARTIELLES D'ADN DU GENE, VECTEURS D'EXPRESSION AVEC LE GENE OU LES SEQUENCES PARTIELLES, SOUCHES DE E. COLI POUR LA PREPARATION DE L-HicDH, L-HicDH ET PROCEDE POUR SON APPLICATION

Country Status (1)

Country Link
WO (1) WO1991003560A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004013332A1 (fr) * 2002-08-01 2004-02-12 Nederlands Instituut Voor Zuivelonderzoek Conversion de substrat

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0103210A2 (fr) * 1982-09-14 1984-03-21 Degussa Aktiengesellschaft Déshydrogénase d'acide L-2-hydroxy-4-méthylpentanoique, procédé pour sa production et son utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0103210A2 (fr) * 1982-09-14 1984-03-21 Degussa Aktiengesellschaft Déshydrogénase d'acide L-2-hydroxy-4-méthylpentanoique, procédé pour sa production et son utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Gene, Band 78, Mai 1989, Elsevier Science Publishers B.V., (Amsterdam, NL), H-P. LERCH et al.: "Cloning, Sequencing and Expression in Escherichia Coli of the D-2-Hydroxyisocaproate Dehydrogenase Gene of Lactobacillus Casei", pages 47-57 siehe den ganzen artikel *
Gene, Band 83, 30. November 1989, Elsevier Science Publishers B.V., (Amsterdam, NL), H-P. LERCH et al.: "Cloning Sequencing and Expression on the L-2-Hydroxyis oicaproate Dehydrogenase-Encoding Gene of Lactobacillus Confusus in Escherichia Coli", pages 263-270 siehe den ganzen artikel *
Proceedings of the 4th European Congress of Biotechnology, Band 2, 1987, Elsevier Science Publishers B.V., (Amsterdam, NL), H. TSAI et al.: "The D- and L-2- Hydroxyisocaproate Dehydrogenases from Lactobacilli are Structurally and Immunologically Unrelated Enzymes", pages 228-231 siehe den ganzen artikel *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004013332A1 (fr) * 2002-08-01 2004-02-12 Nederlands Instituut Voor Zuivelonderzoek Conversion de substrat

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