DK200401262A - Transformation af alkalofile Bacillus-stammer - Google Patents

Transformation af alkalofile Bacillus-stammer Download PDF

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Publication number
DK200401262A
DK200401262A DK200401262A DKPA200401262A DK200401262A DK 200401262 A DK200401262 A DK 200401262A DK 200401262 A DK200401262 A DK 200401262A DK PA200401262 A DKPA200401262 A DK PA200401262A DK 200401262 A DK200401262 A DK 200401262A
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bacillus
host cell
protoplasts
dna
dna sequence
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DK200401262A
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Eekelen Christiaan Albertu Van
Laan Johannes Cornelis Van Der
Mulleners Leonardus Joha Marie
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Genencor Int
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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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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; 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
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    • 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
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

PATENTKRAV 1. Fremgangsmåde til forøgelse af produktionen af et endogent enzym af interesse i en værtscelle af en alkalofil Bacillus-stamme, hvor nævnte fremgangsmåde omfatter: kombination, under tilstedeværelse af et fusogent middel ved alkalisk pH, af en protoplast af nævnte værtscelle og en plasmidkonstruktion omfattende en DNA-sekvens, der koder for nævnte enzym, et replika-tionssystem, som er virksomt i nævnte værtscelle, og et selektionsmarkørgen, hvorved nævnte DNA inkorporeres i nævnte værtscelle,- selektion for nævnte værtsceller indeholdende nævnte DNA i stabilt opretholdt form; og dyrkning af nævnte værtsceller under betingelser, der begunstiger dannelsen af nævnte enzym af interesse. 2. Fremgangsmåde ifølge krav 1, som omfatter, at man: forbehandler nævnte Bacillus-celler med lysozym i et alkalisk medium ved ca. 20°C til ca. 37°C for at danne protoplaster; separerer nævnte protoplaster fra nævnte lysozym; kombinerer nævnte protoplaster med nævnte plasmidkonstruktion under tilstedeværelse af et fusogent middel ved ca. 20°C til 37°C; fortynder nævnte fusogene middel og regenererer nævnte Bacillus-celler; og dyrker nævnte regenererede Bacillus-celler under selektive betingelser. 3. Fremgangsmåde ifølge krav 1 eller krav 2, hvor nævnte plasmidkonstruktion i transkriptionsretningen omfatter en transkriptions- og translations initieringsregion; en DNA-sekvens, der koder for nævnte enzym; og en transkriptions- og transla-tionsterminationsregion, hvor nævnte regulatoriske regioner er virksomme i nævnte værtscelle. 4. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor en utransformeret forældre-celle til nævnte værtscelle er en vildtype- eller mutant alkalofil Bacillus, som danner nævnte serinpro-tease, hvorved endogen produktion af enzymet i værtscellen forøges. 5. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 3, hvor et utransformeret ophav til nævnte værtscelle er en mutant stamme af en vildtype-Bacillus, som ikke danner serinprotease. 6. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor nævnte værtscelle eller vildtype-Bacillus er Bacillus novo art PB92. 7. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor nævnte endogene enzym er en serinprotese. 8. Fremgangsmåde ifølge krav 7, hvor nævnte serinprotease er en høj alkalisk serinprotease. 9. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor nævnte enzym er en høj alkalisk serinprotease med mindst 70% homologi med amino-syresekvensen:
HjN-A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V-L-D-T-G-I-S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P-S-T-Q-D-G-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-1-G-V-L-G-V-A-P-N-A-E-L-Y-A-V-K-V-L-G-A-S-G-S-G-S-V-S-S-1 -A-Q-G-L-E-W-A-G-N-N-G-M-H-V-A-N-L-S-It-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V-A-A-S-G-N-S-G-A-G-S-I-S-Y-P-A-R-Y-A-N-A-M-A-V-G-A-T-D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I -V-A-P-G-V-N-V-Q-S-T-Y-P-G-S-T-Y-A-S-L-N-G-T-S-M-A-T-F-H-V-A-G-A-A-A-L-V-K-Q-K-N-P-S-W-S-N-V-Q-I-R-N-H-L-K-N-T-A-T-S-L-G-S-T-N-L-Y-G-S-G-L-V-N-A-E-A-A-T-R-OOOH. 10. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor nævnte DNA er stabilt integreret i nævnte værtscellegenom. 11. Fremgangsmåde ifølge krav 10, hvor mindst to kopier af nævnte DNA er stabilt integreret i nævnte værtscellegenom. 12. Fremgangsmåde ifølge krav 10, hvor to kopier af proteasegenet sidder i tandem i værtscellekromosomet, adskilt af plasmidsekvenser. 13. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor markørgenet er et antibiotisk resistensgen.
DK200401262A 1987-02-27 2004-08-20 Transformation af alkalofile Bacillus-stammer DK175852B1 (da)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK200401262A DK175852B1 (da) 1987-02-27 2004-08-20 Transformation af alkalofile Bacillus-stammer

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP87200358 1987-02-27
DK198805939A DK175738B1 (da) 1987-02-27 1988-10-26 Molekylær kloning og ekspression af gener, der koder for proteolytiske enzymer
DK200401262A DK175852B1 (da) 1987-02-27 2004-08-20 Transformation af alkalofile Bacillus-stammer

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DK200401262A true DK200401262A (da) 2004-08-20
DK175852B1 DK175852B1 (da) 2005-04-04

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DK198805939A DK175738B1 (da) 1987-02-27 1988-10-26 Molekylær kloning og ekspression af gener, der koder for proteolytiske enzymer
DK200401262A DK175852B1 (da) 1987-02-27 2004-08-20 Transformation af alkalofile Bacillus-stammer

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US (1) US5217878A (da)
JP (1) JP3026567B2 (da)
KR (1) KR0127560B1 (da)
CN (1) CN88101681A (da)
AT (2) ATE181110T1 (da)
AU (1) AU620026B2 (da)
BG (1) BG49049A3 (da)
BR (1) BR8805647A (da)
CA (1) CA1339100C (da)
DE (2) DE3856331T2 (da)
DK (2) DK175738B1 (da)
FI (3) FI105483B (da)
HU (1) HU213220B (da)
LT (1) LT4000B (da)
LV (1) LV10307B (da)
NO (1) NO884423D0 (da)
PT (1) PT86864B (da)
RU (1) RU2023723C1 (da)
WO (1) WO1988006624A2 (da)

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RU2023723C1 (ru) 1994-11-30
FI105483B (fi) 2000-08-31
DK593988A (da) 1988-10-26
FI106726B (fi) 2001-03-30
DE3856339T2 (de) 1999-11-04
JP3026567B2 (ja) 2000-03-27
BR8805647A (pt) 1989-10-31
FI884904L (fi) 1988-10-24
FI925413L (fi) 1992-11-27
AU620026B2 (en) 1992-02-13
AU1398088A (en) 1988-09-26
BG49049A3 (bg) 1991-07-15
LV10307B (en) 1995-08-20
WO1988006624A2 (en) 1988-09-07
DK175852B1 (da) 2005-04-04
ATE181110T1 (de) 1999-06-15
FI119028B (fi) 2008-06-30
CN88101681A (zh) 1988-11-09
HU213220B (en) 1997-03-28
PT86864B (pt) 1992-05-29
FI925413A0 (fi) 1992-11-27
LT4000B (en) 1996-06-25
JPH01502876A (ja) 1989-10-05
NO884423L (no) 1988-10-05
US5217878A (en) 1993-06-08
LV10307A (lv) 1994-10-20
DE3856339D1 (de) 1999-07-15
DK593988D0 (da) 1988-10-26
NO884423D0 (no) 1988-10-05
CA1339100C (en) 1997-07-29
DK175738B1 (da) 2005-02-07
PT86864A (pt) 1988-03-01
KR890700665A (ko) 1989-04-26
FI20001152L (fi) 2000-05-15
FI884904A0 (fi) 1988-10-24
DE3856331T2 (de) 1999-10-14
DE3856331D1 (de) 1999-06-17
ATE180016T1 (de) 1999-05-15
LTIP1820A (en) 1995-08-25
KR0127560B1 (ko) 1997-12-29
WO1988006624A3 (en) 1988-11-17

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