KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Cloning of a $\Delta5$ desaturase from Thraustochytrium sp. 26185 and Functional Expression in Pichia Pastoris

Thraustochytrium sp. 26185 균주에서의 $\Delta5$ desaturase 유전자 클로닝 및 Pichia pastoris 내에서의 기능적 발현

  • Chung Tae-Ho (Department of Biological and Chemical Engineering, College of Engineering, Inha University) ;
  • Lee Su-Jin (Department of Biological and Chemical Engineering, College of Engineering, Inha University) ;
  • Oh Hyo-Jeong (Department of Biological and Chemical Engineering, College of Engineering, Inha University) ;
  • Kim Geun-Joong (Department of Biological and Chemical Engineering, College of Engineering, Inha University) ;
  • Hur Byung-Ki (Department of Biological and Chemical Engineering, College of Engineering, Inha University)
  • 정태호 (인하대학교 공과대학 생물공학과 생물산업기술연구소) ;
  • 이수진 (인하대학교 공과대학 생물공학과 생물산업기술연구소) ;
  • 오효정 (인하대학교 공과대학 생물공학과 생물산업기술연구소) ;
  • 김근중 (인하대학교 공과대학 생물공학과 생물산업기술연구소) ;
  • 허병기 (인하대학교 공과대학 생물공학과 생물산업기술연구소)
  • Published : 2005.04.01
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Abstract

Polyunsaturated fatty acids, that is PUFAs, are important constituents of membranes particularly found in the retina and central nervous system. In microorganism-based PUFAs biosynthesis, the genus Thraustochytrids is well evaluated for their potential as a promising candidate in the practical production of PUFAs, such as AA and DHA. In this study, we attempted to optimize a method of total nucleic acid extraction from this microorganism as a preliminary experiment. Using the extracted nucleic acid and degenerated primers for direct PCR, we isolated a $\Delta5$ desaturase gene that contained 1320-nucleotide and encoded 439 amino acids. This gene exhibited an expected function, when expressed in P. pastoris in the presence of appropriate exogenous substrate, as an evidence for $\Delta5$ desaturase activity (conversion of DGLA to AA). These results and information could provide a basis for the construction of engineered strains suitable for the practical production of PUFAs.

불포화지방산의 생합성능이 뛰어나다고 알려진 Thraustochytrids 균주 중 26185를 대상으로 arachidonic acid (AA C20:4 n-6)를 포함하는 PUFA 생산에 관여하는 유전자의 하나로서 key enzyme인 desaturase를 보다 효율적이고 안정적인 방법으로 cloning한 후 결과물을 분석하였다. 이를 위해 실험균주인 26185 균주를 대상으로 효율적인 전체 nucleic acids 추출법을 개발하였으며 관련된 기법의 활용을 통해 일반적으로 활용이 가능한 효과적이 방법임을 확인하였다. 확보된 유전체를 통한 direct PCR의 결과물을 기존에 알려진 cDNA 합성방법에 의한 결과물과 비교하였을 때 동일한 결과물임을 확인하였다. 이는 Thraustochytrids 관련 균주로부터 지방산 생합성에 관련된 효소군을 탐색하는 방법이 cDNA 합성방법에 의하지 않고 보다 직접적으로 진행될 수 있음을 제안할 수 있는 하나의 결과물로 생각되어진다. 획득된 유전자를 같은 진핵세포인 P. pastoris를 숙주로 하여 유전자를 도입하고 활성을 재현성 있게 규명함으로써 인공진화 혹은 재조합균체의 개발이 가능하다는 사실을 부분적으로 제시할 수 있었다.

Keywords

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