Structure and function of strong promoters in bacillus subtilis

Abstract

For the purpose of Bacillus subtilis as host cells for genetically engineered genes to over-produce useful products extracellularly, promoters which function strongly in B. subtilis were tried to develop in this study. The native promoter of an endoglucanase gene of B. subtilis origin was removed and the remaining structural gene(ENG) was used as a model. To the ENG, strong promoters such as tac promoter (Ptac) and promoters isolated from B. subtilis 168 were fused and their functions in B subtilis were observed. These promoters were then modified by altering nucleotide sequences and their responses on transcriptional efficiency were analyzed to find general requirements on the structure of a strong promoter in B. subtilis. The ENG structural gene contained in pBSU1028 was isolated and fused to Ptac of pKK223-3 to make a new endoglucanase gene Ptac-ENG in which the native promoter of B. subtilis endoglucanase gene was replaced by Ptac. The newly constructed plasmid Ptac-ENG is designated pBSKTA. With pBSKTA, E. coli JM103 was transformed to make a new transformat E. coli JM103 (pBSKTA). The new endoglucanase gene under control of Ptac in E. coli JM103 (pBskta) expressed very well in LB medium when induced by IPTG and produced endoglucanase up to 23\% of the total cell protein. However, most of the endoglucanase produced was remained in the cell as inclusion body having no enzyme activity. The Ptac-ENG gene in pBSKTA was transferred to pUB110, a Bacillus vector, and with the resulting plasmid pBSKTAU, B. subtilis RM125 was transformed to make a new transformant B. subtilis RM125 (pBSKTAU). The Ptac-ENG gene expressed in B. subtilis RM125 (pBSKTAU) and produced endoglucanase indicating that the Ptac originally developed to use in E. coli can function in B. subtilis also. The production of endoglucanase by B. subtilis RM125 (pBSKTAU) was however, one third of that by B. subtilis RM125(pCK98) at the peak. The plasmid pCK98 contains ENG structural gene with native p...