출아효모에서 재조합 neoagarobiose hydrolyase의 생산을 위한 최적 발현시스템
Optimal Expression System for Production of Recombinant Neoagarobiose Hydrolyase in Saccharomyces cerevisiae
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초록
본 연구에서는 Saccharomyces cerevisiae를 이용해서neoagarobiose hydrolase (NABH)를 효율적으로 생산하기위한 NABH558 유전자 발현시스템을 구축하였다. ADH1 promoter와 GAL10 promoter 하류에 NABH558 유전자를가진 pAMFα-NABH plasmid와 pGMFα-NABH plasmid 는 S. cerevisiae 2805 균주에 형질전환되었다. 2805/ pAMFα-NABH 균주는 YPD (2% dextrose) 배지에서 가장높은 NABH 효소 활성(0.069 unit/ml/DCW)을 보였고, 2805/pGMFα-NABH 균주의 경우는 배지의 조성과 상관없이 비슷한 수준의 NABH 활성(0.02−0.027 unit/ml/DCW)을 보였다. RT-PCR을 통한 NABH558 유전자의 transcription level은 NABH 활성 증가에 따라 비슷한 수준으로 증가되었음을 확인할 수 있었다. 또한 재조합균주에서 생산된 NABH 는 agarose를 galactose와 AHG로 분해하였다. 따라서NABH558 유전자의 발현에는 ADH1 promoter를 사용하는것이 더 효율적이며 GAL10 promoter와 비교해서 최대 3배정도 높은 활성의 재조합 NABH를 생산할 수 있음을 알 수있었다.
Abstract
In this study, the NABH558 gene expression system was constructed to efficiently produce neoagarobiose hydrolase (NABH) in Saccharomyces cerevisiae strain. The ADH1 and GAL10 promoters of the pAMFα- NABH and pGMFα-NABH plasmids were examined to determine the suitable promoter for the NABH558 gene expression, respectively. The effect of promoter and carbon sources on NABH558 gene expression was investigated by transforming each plasmid into the S. cerevisiae 2805 strain. The NABH activity in the 2805/ pAMFα-NABH strain was 0.069 unit/ml/DCW in YPD medium, whereas that in the 2805/pGMFα-NABH strain was similar (0.02−0.027 unit/ml/DCW) irrespective of the medium composition. The higher NABH activity in the YPD medium was due to the increased NABH558 gene transcription. NABH produced in the recombinant strains could degrade agarose to galactose and AHG. This indicated that ADH1 promoter was a more optimal promoter for the expression of NABH558 gene than the GAL10 promoter. The NABH activity induced by the ADH1 promoter was about 3-fold higher than that induced by the GAL10 promoter.
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