枯草芽孢杆菌P_(spovG)启动子改造与表征

Engineering and Characterization of Bacillus subtilis P_(spovG) Promoter

枯草芽孢杆菌是重要的工业生产菌株,广泛应用于外源蛋白质的表达。目前在枯草芽孢杆菌中常见组成型启动子中,P_(spovG)作为高强度组成型启动子在表达外源蛋白质时,具有表达量高、稳定性好和适用范围广等优势。作者首先对P_(spovG)上游序列截短,发现将启动子上游序列由原来的251个碱基缩短为26个,仍能保持启动子强度不降低,并确定了紧邻-35元件上游的3个碱基“AGC”为影响启动子强度的关键碱基。其次对启动子的上游A-T富含区以及spacer区域的G-C富含区进行随机突变,证明了上游激活序列中A-T碱基的排列对启动子强度有重要影响,并得到了强度提高的突变体。最后通过将不同表达时期启动子的关键调控序列分别插入突变体的上下游,得到了启动子强度为原始启动子135.1%的新型启动子SP_(spovG)-P_(lytR)。本研究为枯草芽孢杆菌表达系统开发新型强组成型启动子提供了重要的参考。

Bacillus subtilis is an important industrial production strain,widely used in the expression of foreign proteins.At present,among the common constitutive promoters in Bacillus subtilis,P_(spovG),as a high-strength constitutive promoter,has the advantages of high expression,good stability and wide application when expressing foreign proteins.This study first truncated the upstream sequence of P_(spovG),and the strength of the promoter was still maintained when the upstream sequence of promoter was shortened form the original 251 bases to 26.And the three bases'AGC'adjacent to the upstream of element-35 was identified as the key bases affecting the promoter strength.Secondly,random mutations of the upstream A-T rich region of promoter and the G-C rich region of spacer region were conducted,which proved that the arrangement of the A-T bases in the upstream activation sequence had an important influence on the promoter strength,and a mutant with increased strength was obtained.Finally,a novel promoter SP_(spovG)-P_(lytR) with a promoter strength of 135.1%of the original promoter was obtained by inserting the key regulatory sequences of promoters at different expression periods into the upstream and downstream of mutant.This study provides an important reference for the development of new strong constitutive promoters for the Bacillus subtilis expression system.