Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of pSJE6c, an Expression Vector for Kimchi Lactic Acid Bacteria, and Heterologous Gene Expression Using the Vector

김치유산균용 발현벡터 pSJE6c 개발과 이를 이용한 외래 유전자 발현

  • Lee, Kang-Wook (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Park, Ji-Yeong (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Lee, Ji-Yeon (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Lee, Hwang-A (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Baek, Chang-Un (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Jo, Hyeon-Deok (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Kim, Joo-Yeon (Division of Applied Life Science (BK21 program), Graduate School) ;
  • Kwon, Gun-Hee (Institute of Agriculture & Life Science, Gyeongsang National University) ;
  • Chun, Ji_Yeon (Department of Food Science and Technology, Sunchon National University) ;
  • Kim, Jeong-Hwan (Division of Applied Life Science (BK21 program), Graduate School)
  • 이강욱 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 박지영 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 이지연 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 이황아 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 백창운 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 조현덕 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 김주연 (경상대학교 대학원 응용생명과학부(BK21 program)) ;
  • 권건희 (경상대학교 농업생명과학원) ;
  • 천지연 (순천대학교 식품공학과) ;
  • 김정환 (경상대학교 대학원 응용생명과학부(BK21 program))
  • Published : 2009.12.28
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Abstract

Development of expression vectors is important for the basic and applied researches on kimchi LAB (lactic acid bacteria). An expression vector, pSJE6c was constructed by inserting P6C promoter sequence from Lactococcus lactis into pSJE, a shuttle vector for E. coli and Leuconostoc species. To test the efficiency of pSJE6c, aga ($\alpha$-galactosidase) and lacZ ($\beta$-galactosidase) genes were expressed in Lactobacillus brevis 2.14. Compared to the pSJE, expression levels of both genes were increased, indicating P6C promoter was better than indigenous promoters. Enzyme activities of L. brevis cells harboring pSJE6caga (pSJE6c with aga) or pSJE6Z (pSJE6c with lacZ) were 1.5-2 fold higher than those with pSJEaga (pSJE with aga) or pSJEZ (pSJE with lacZ). More RNA transcripts were detected in cells harboring pSJE6c based recombinant plasmid. The results indicated that heterologous gene expressions in kimchi LAB could be improved significantly by use of efficient expression vectors.

본 연구실에서 개발한, 김치에서 분리한 Leu. mesenteroides SY2 유래 pFMBL1 을 바탕으로 구축한 셔틀벡터인, pSJE[7]를 외래유전자 발현에 적합하게 개량한 발현벡터를 구축하였다. Lactococcus lactis LM0230에서 분리한 프로모터 P6C를 pSJE에 도입하였다. P6C 염기서열을 지닌 oligonucleotide 쌍을 따로 제조한 후 annealing을 통해 짧은 DNA 단편을 얻어서 제한효소 처리후 pSJE에 도입하여 pSJE6c를 구축하였다. PSJE6c 효능 검증을 위해서 외래 유전자인 aga와 lacZ를 각각 pSJE6c에 도입하였다. P6C 프로모터와 비교를 위해 고유 프로모터를 지닌 유전자들도 각각 pSJE에 도입하였다. 재조합 plasmid들을 electroporation 방법으로 Lactobacillus brevis 2.14 균주에 도입하고 재조합균주들의 생육곡선과 효소역가 그리고 slot blot으로 전사체 농도를 측정하였다. 결과를 보면 PSJE6c에 클로닝 된 유전자들이 pSJE상의 유전자보다 효소역가들이 약 1.5배에서 2배 정도로 높았다. 전사체 농도 측정 결과도 pSJE6c 들에서 더 많은 전사체가 생성됨을 보여주었다. 이상 결과들은 효율적인 발현벡터들의 사용을 통해서 김치유산균에서 외래유전자 발현 효율을 높일 수 있음을 보여준다.

Keywords

References

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