蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段，该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达，核黄素产量从39．5mg／L增加到61．7mg/L.

Cloning the riboflavin operon of B.cereus ATCC14579 and expression in B.subtilis

A 4 5kb DNA fragment containing the whole riboflavin operon was detected from B cereus ATCC 14579 genome in a sequence similarity search against B cereus genome sequence (database) with the B subtilis riboflavin operon as the query sequence The deduced amino acid sequence of the riboflavin operon of B cereus ATCC 14579 exhibited 99% similarity with that of the riboflavin operon from B subtilis This fragment was cloned into B subtilis E coli shuttle vector pHP13M Expression analysis showed that the riboflavin operon of B cereus ATCC 14579 was operative in both E coli and B subtilis However, its expression in B subtilis did not result in overproduction of riboflavin The original promoter of the operon was replaced by the sac B promoter from B subtilis by a PCR method for its better expression This substitution led to enhanced production of riboflavin under the fermentation condition used in this study The yield of riboflavin increased from 39 5 mg/L to 61 7 mg/L