Abstract
Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.
Strong promoter로 밝혀진 alginate lyase 유전자의 promoter 부위에 대한 특성을 검토하기 위해 alginate lyase 유전자의 46개 N-terminal amino acid가 포함된 promoter 부분과, 같은 균으로부터 분리한 $\beta$-agarase의 유전자를 연결시켜 agarase의 activity를 평판배지상에서 보다 쉽게 확인하는 방법으로 promoter의 활성을 측정한 결과 alginate lyase 유전자 promoter에 의해서 $\beta$-agarase 유전자의 대량발현이 유도되고 있었으며 glucose의 존재하에서 $\beta$-agarase 유전자 발현이 일어나지 않는 catabolite repression 양상을 나타내고 있다. PCR로써 alginate lyase의 46개 N-terminal amino acid 부분이 순차적으로 제거된 plasmid를 제조하여 대량발현을 조사한 결과 46개의 아미노산이 제거된 후에도 $\beta$-agarase의 활성에는 변화가 없어 46개의 N-말단이 정상적인 상태에서 발현에는 영향을 미치고 있지 않음을 확인할 수 있었다. 또한 alginate lyase 유전자의 promoter region에 존재하는 가능한 2개의 promoter consensus sequence PI, PII를 subcloning한 결과 promoter PII만이 존재할 때도 대량발현이 유도되고 있음을 확인할 수 있었으며 동시에 glucose가 존재할 때 catabolite repression이 역시 나타나고 있어 이 부분이 발현 및 glucose에 의한 regulation에 매우 중요하게 작용하는 부분이라는 것을 확인할 수 있었다.