EP0652958A1 - Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas - Google Patents

Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas

Info

Publication number
EP0652958A1
EP0652958A1 EP93917663A EP93917663A EP0652958A1 EP 0652958 A1 EP0652958 A1 EP 0652958A1 EP 93917663 A EP93917663 A EP 93917663A EP 93917663 A EP93917663 A EP 93917663A EP 0652958 A1 EP0652958 A1 EP 0652958A1
Authority
EP
European Patent Office
Prior art keywords
lipase
pseudomonas
gene
leu
ala
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.)
Withdrawn
Application number
EP93917663A
Other languages
German (de)
English (en)
Inventor
Maria Mathilde Josephina Cox
Gijsbert Gerritse
Wilhelmus Johannes Quax
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.)
Danisco US Inc
Original Assignee
Gist Brocades NV
Genencor International Inc
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 Gist Brocades NV, Genencor International Inc filed Critical Gist Brocades NV
Priority to EP93917663A priority Critical patent/EP0652958A1/fr
Publication of EP0652958A1 publication Critical patent/EP0652958A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention describes the cloning and expression of a lipase gene in combination with a lipase modulator gene, both obtained from a class I Pseudomonas species, in an homologous class I Pseudomonas species.
  • Lipases are enzymes capable of hydrolyzing lipids they are utilized in a wide range of applications such as fats and oil processing, detergents, diagnostic reagents etc.
  • Extracellular lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are produced by a wide variety of micro- organisms. Suitable microbial lipases have for example been disclosed in U.S. Patent No. 3,950,277, these lipases were obtained from such diverse microorganisms as Pseudomonas. Asper ⁇ illus. Pneumococcus. Staphylococcus. ycobacterium tuberculosis. Mycotorula lipolytica and Sclerotinia. It has turned out that especially Pseudomonas lipases have favourable characteristics for the desired applications. Pseudomonas species have therefore been extensively used for obtaining lipases.
  • EP 331376 describes the cloning and expression of a lipase gene obtained from Pseudomonas cepacia in P. cepacia. It was found that no expression could be obtained when a second gene located downstream of the lipase gene was deleted. This gene was therefore reported to be essential for lipase production.
  • EP 464922 reports the cloning and expression of a lipase gene together with a gene encoding a protein reported to have a lipase-specific stabilizing/translocation function. The genes are obtained from Pseudomonas qlumae and expression is preferably in heterologous systems. The stabilising protein is reported to differ greatly from the gene described in EP 331376 and therefor assumed to have a different function.
  • WO 91/00908 reports the expression in a heterologous host of the lipase gene and the lipase modulator gene obtained from P. cepacia.
  • Lipase modulator genes are reported to be essential for obtaining lipase production, however for an extensively investigated representative of class I Pseudomonas species: Pseudomonas fraqi such a gene was not found. Another class I Pseudomonas lipase gene was described in EP 334462.
  • EP 334462 reports the cloning and expression of the lipase gene from Pseudomonas pseudoalcaliqenes in E. coli it can be concluded that for heterologous lipase production the lipase modulator gene was not essential.
  • the classification of Pseudomonas species is based on DNA-rRNA and DNA-DNA hybridization studies as reported by Palleroni et al.
  • the present invention discloses a lipase modulator gene and the corresponding protein obtained from a class I Pseudomonas species.
  • the present invention also discloses Pseudomonas strains which have been transformed with a DNA sequence encoding a lipase and a sequence encoding a lipase modulator gene. These strains are preferably class I Pseudomonas strains and more preferably Pseudomonas pseudoalcaliqenes strains. The present invention further discloses a method for obtaining such transformed strains.
  • the invention further discloses a vector derived from pJRD215 and which is segregationally stable in Pseudomonas. A method for obtaining such a vector is also disclosed.
  • Km r gene encoding neo ycin resistance of Tn5.
  • lip gene encoding Ml lipase. Furthermore a number of restriction sites are indicated. - 4 -
  • Figure 2 Sequence of pJRD215 (derived from Davison et al.
  • Km r gene encoding neomycin resistance of Tn5.
  • lip gene encoding Ml lipase. Due to the deletion plasmid PlA ⁇ is about 900 bp smaller than P1A, also several restriction sites are missing.
  • Figure 4 Construction and restriction map of plasmid PIB.
  • Plasmid pTMPvl ⁇ A was described in EP 334462.
  • lip gene encoding Ml lipase, location indicated by an arrow Tc r : gene encoding tetracyclin resistance.
  • Km r gene encoding neomycin resistance of Tn5.
  • lip gene encoding Ml lipase.
  • lim gene encoding the Ml lipase modulator protein.
  • Figure 6 Restriction map of plasmid P24B.
  • lip gene encoding Ml lipase, location indicated by an arrow lim: gene encoding the Ml lipase modulator protein, location also indicated by an arrow.
  • the recombinant DNA of the present invention is obtained by digestion of chromosomal DNA obtained from a strain of a Pseudomonas class I species.
  • Representatives of class I Pseudomonas species are: Pseudomonas alcaligenes. Pseudomonas pseudoalcaligenes. Pseudomonas stutzeri. Pseudomonas aeruginosa. and Pseudomonas mendocina.
  • the chromosomal DNA is isolated using standard procedures as disclosed for example in Maniatis et al. Molecular cloning. Cold Spring Harbor Press, 1982 and 1989. I suitable digest is made and the fragments are cloned in a vector which is subsequently used to transform an E. coli. Selection is made on the basis of the presence of the lipase gene this can be performed using hybridization if suitable probes are available. Alternatively it is possible to use an expression vector in which case it becomes possible to select for the presence of the desired genes using a suitable assay such as halo formation when lipase is screened for. Furthermore expression can also be monitored using immunological detection of the protein when suitable antibodies are available.
  • lipase modulator gene is necessary for lipase production the chromosomal copy of the gene is sufficient to allow an increase of 2000%. Only at this point introduction of additional modulation gene copies will result in higher lipase production.
  • Lipase and lipase modulator gene sequences were compared using computer analysis.
  • a mole ⁇ cular enzyme screening assay as described in EP 334462. From this work it was concluded that Pseudomonas species belonging to the same RNA homology group as P. pseudoalcaligenes show a rather strong homology, whereas Pseudomonas species belonging to a different RNA homology group (Palleroni, 1973) and other bacterial species show no hybridization at all. A correlation between both methods was well established, which makes it possible to determine homology with uncharacterized lipase genes.
  • TCCCCTCGGT AACATCCCCT AGGTAATAGC AGAGCCCTTG CCGGCGCTGG CTTTCGTCAC 120
  • Met Asn Asn Lys Lys Thr Leu Leu Ala Leu Cys lie Gly Ser Ser Leu -24 -20 -15 -10
  • Gin Arg Thr Leu Asp Asp Ala Pro Ala Ala Pro Pro Leu Ala Ala Glu 50 55 60 lie Ala Pro Leu Pro Pro Ser Phe Ala Gly Thr Gin Val Asp Gly Gin 65 70 75 80

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne le clonage et l'expression d'un gène modulateur de lipase obtenu à partir d'une espèce de Pseudomonas de classe I. On a découvert que le produit d'expression du gène modulateur donne lieu à une augmentation considérable de la production de lipase, notamment lors d'une expression homologue. L'invention concerne également un procédé d'isolement d'un gène modulateur de lipase de classe I, un gène modulateur isolé ainsi qu'un Pseudomonas de classe I transformé à l'aide dudit gène. Enfin, l'invention concerne un dérivé de plasmide pJRD215 stable séparément dans Pseudomonas.
EP93917663A 1992-07-23 1993-07-23 Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas Withdrawn EP0652958A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP93917663A EP0652958A1 (fr) 1992-07-23 1993-07-23 Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP92202281 1992-07-23
EP92202281 1992-07-23
PCT/EP1993/001995 WO1994002617A2 (fr) 1992-07-23 1993-07-23 Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas
EP93917663A EP0652958A1 (fr) 1992-07-23 1993-07-23 Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas

Publications (1)

Publication Number Publication Date
EP0652958A1 true EP0652958A1 (fr) 1995-05-17

Family

ID=8210805

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93917663A Withdrawn EP0652958A1 (fr) 1992-07-23 1993-07-23 Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas

Country Status (3)

Country Link
EP (1) EP0652958A1 (fr)
JP (1) JPH07509129A (fr)
WO (1) WO1994002617A2 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0334462B2 (fr) † 1988-03-25 2002-04-24 Genencor International, Inc. Clonage et expression moléculaire de gènes codant pour enzymes lipolytiques
CA2189441C (fr) * 1994-05-04 2009-06-30 Wolfgang Aehle Lipases a resistance aux tensioactifs amelioree
BE1008998A3 (fr) * 1994-10-14 1996-10-01 Solvay Lipase, microorganisme la produisant, procede de preparation de cette lipase et utilisations de celle-ci.
BE1008783A3 (fr) * 1994-10-14 1996-08-06 Solvay Lipase, microorganisme la produisant, procede de preparation de cette lipase et utilisations de celle-ci.
JPH08228778A (ja) * 1995-02-27 1996-09-10 Showa Denko Kk 新規なリパーゼ遺伝子及びそれを用いたリパーゼの製造方法
BE1009312A3 (fr) * 1995-05-05 1997-02-04 Solvay Compositions detergentes.
BE1009650A5 (fr) * 1995-10-12 1997-06-03 Genencor Int Systeme d'expression, vecteur et cellule transformee par ce vecteur.
US7288400B2 (en) 1996-02-16 2007-10-30 Verenium Corporation Nucleic acids encoding esterases and methods of making and using them
US5942430A (en) * 1996-02-16 1999-08-24 Diversa Corporation Esterases
WO1998006836A2 (fr) * 1996-08-16 1998-02-19 Genencor International, Inc. Systeme d'expression pour niveaux d'expression modifies
US20140031272A1 (en) 2011-04-08 2014-01-30 Danisco Us Inc. Compositions

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3079276B2 (ja) * 1988-02-28 2000-08-21 天野製薬株式会社 組換え体ｄｎａ、それを含むシュードモナス属菌及びそれを用いたリパーゼの製造法
EP0334462B2 (fr) * 1988-03-25 2002-04-24 Genencor International, Inc. Clonage et expression moléculaire de gènes codant pour enzymes lipolytiques
DK336889D0 (da) * 1989-07-07 1989-07-07 Novo Nordisk As Polypeptider
EP0464922A1 (fr) * 1990-07-06 1992-01-08 Unilever N.V. Préparation de lipase active de pseudomonas glumae à partir d'hôtes homologues ou hétérologues

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J. HERMANS ET AL.: "Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range Gram-negative cosmid vector pJRD215", MOLECULAR MICROBIOLOGY, vol. 5, no. 6, 1991, pages 1561 - 1566, XP001002547 *
JOHN DAVISON ET AL.: "Vectors with restriction site banks V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites", GENE, vol. 51, 1987, AMSTERDAM NL, pages 275 - 280, XP002168737 *
P.K.R. KUMAR ET AL.: "Strategies for improving plasmid modofied stability in genetically modified bacteria in bioreactors", TRENDS IN BIOTECHNOLOGY, vol. 9, no. 8, 1991, pages 279 - 284, XP000233077 *
See also references of WO9402617A3 *

Also Published As

Publication number Publication date
WO1994002617A3 (fr) 1994-06-09
WO1994002617A2 (fr) 1994-02-03
JPH07509129A (ja) 1995-10-12

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