• 제목/요약/키워드: multicopy gene expression system

검색결과 4건 처리시간 0.014초

Control of Acetate Production Rate in Escherichia coli by Regulating Expression of Single-Copy pta Using $lacI^Q$ in Multicopy Plasmid

  • Lee, Sun-Gu;Liao, James C
    • Journal of Microbiology and Biotechnology
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    • 제18권2호
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    • pp.334-337
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    • 2008
  • A tightly regulated gene expression system composed of a single-copy target gene under the control of a lac promoter derivative and lacI gene in a multicopy plasmid is proposed, and its ability to control the flux of a metabolic pathway is demonstrated. A model system to control the flux of acetyl-CoA to acetyl phosphate was constructed by integrating pta, a gene encoding phosphotransacetylase, under a tac promoter into the chromosome of E. coli with a pta-negative background and transforming a multicopy plasmid containing the $lacI^Q$ gene into the strain. The production rate of acetate was shown to be tightly controlled when varying the concentration of the inducer (IPTG) in he model system.

Expression of the Aspergillus niger var. awamori Phytase Gene in Pichia pastoris, and Comparison of Biological Properties

  • CHOI, JAE-MUN;DOO-SANG KIM;MOON-SICK YANG;HYUNG-RAK KIM;JAE-HO KIM
    • Journal of Microbiology and Biotechnology
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    • 제11권6호
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    • pp.1066-1070
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    • 2001
  • The PhyA gene, encoding myo-inositol hexakisphosphate phosphohydrolase in Aspergillus niger var. awamori (wild-type), was cloned and sequenced. The cDNA was overexpressed by a multicopy gene expression system in Pichia pastoris KM71. Recombinant, wild-type and commercial phytase from Aspergilus ficuum NRRL 3135 (Natuphos) were purified. The PhyA gene of Aspergillus niger var awamori showed perfect homology to the phytase of Aspergillus ficcum and $97\%$ homology to A. niger var awamori (L02421). Wild-type phytase was highly glycosylated and more thermostable than the other two, while deglycosylated farms of three phytases showed identical molecular weight, 507 kDa. After heating at $80^{\circ}C$, wild-type, commercial, and recombinant phytases retained $57\%, 32%,\;and\;8\%$ of their original activities, respectively. In conclusion, glycosylation plays a key role in the thermostability of phytase and its enzymatic characterization.

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Simultaneous and Sequential Integration by Cre/loxP Site-Specific Recombination in Saccharomyces cerevisiae

  • Choi, Ho-Jung;Kim, Yeon-Hee
    • Journal of Microbiology and Biotechnology
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    • 제28권5호
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    • pp.826-830
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    • 2018
  • A Cre/loxP-${\delta}$-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae. To allow repeated integrations, the reusable Candida glabrata MARKER (CgMARKER) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and ${\beta}$-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

약독화 Salmonella typhimurium 생백신 균주에서 Bordetella pertussis 의 filamentous hemagglutinin(F HA) (Expression of recombinant Bordetella pertussis filamentous hemagglutinin (FHA) antigen in Live Attenuated Salmonella typhimurium Vaccine Strain)

  • 강호영
    • 생명과학회지
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    • 제11권4호
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    • pp.385-391
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    • 2001
  • Filamentous hemagglutinin (FHA) is considered as an essential immunogenic component for incorporation into acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. Classically, antipertussis vaccination has employed an intramuscular route. An alternative approach to stimulate mucosal and systemic immune responses is oral immunization with recombinant live vaccine carrier strains of Salmonella typhimurium. An attenuated live Salmonella vaccine sgrain($\Delta$cya $\Delta$crp) expressing recombinant FHA(rFHA) was developed. Stable expressionof rFHA was achieved by the use of balanced-lethal vector-host system. which employs an asd deletion in the host chromosome to impose in obligate requirement for diaminopimelic acid. The chromosomal $\Delta$asd mutation was complemented by a plasmid vector possessing the asd$^{+}$ gene. A 3 kb DNA fragment encoding immuno dominant regionof FHA was subcloned in-frame downstream to the ATG translation initiation codon in the multicopy Asd$^{+}$ pYA3341 vector to create pYA3457. Salmonella vaccine harboring pYA3457 expressed approximately 105kDa rFHA protein. The 100% maintenance of [YA3457 in vaccine strain was confirmed by stability examinations. Additionally, a recombinant plasmid pYA3458 was constructed to overpress His(8X)-tagged rFHA in Essherichia coli. His-tagged rFHA was purified from the E. coli strain harboring pYA3458 using Ni$^{2+}$-NTA affinity purification system.>$^{2+}$-NTA affinity purification system.

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