基于全局转录工程促进肺炎克雷伯氏菌吡咯喹啉醌的生物合成  

Enhanced production of pyrroloquinoline quinone in Klebsiella pneumoniae via global transcription machinery engineering

作　　者：张军静 田平芳[1] 

机构地区：[1]北京化工大学生命科学与技术学院北京市生物加工过程重点实验室,北京100029

出　　处：《北京化工大学学报（自然科学版）》2014年第3期92-96,共5页Journal of Beijing University of Chemical Technology(Natural Science Edition)

基　　金：国家自然科学基金(21076013/21276014)

摘　　要：用易错PCR构建肺炎克雷伯氏菌(Klebsiella pneumoniae)RNA聚合酶rpoD基因的突变文库,将其与载体连接后转化肺炎克雷伯氏菌,从1500个阳性克隆中筛选出一株吡咯喹啉醌(PQQ)生产菌。发酵试验表明,该工程菌的PQQ产量为76mg/mL,比携带未突变rpoD基因的对照菌提高了10%。测序发现rpoD基因在核酸和蛋白质水平的突变率分别达到2.5%和0.5%。二级结构预测发现α螺旋、β转角和无规则卷曲数目均发生了变化,推测由此改变了σ因子与启动子的亲和程度,从而导致了PQQ基因的转录以及K.pneumoniae代谢流量再分配,使得PQQ得到更高效表达。The sigma factor coding gene rpoD in Klebsiella pneumoniae was cloned by the polymerase chain reaction( PCR) and its mutant library was constructed using the error-prone PCR method. Subsequent ligation to an expression vector and transformation into K. pneumoniae DSM 2026 led to a large number of positive clones. Of these, one recombinant strain produced 76 mg / mL of pyrroloquinoline quinone( PQQ),which is 10% higher than the control that expressed the unmutilated rpoD gene. DNA sequencing showed that the mutation rates of rpoD at nucleotide and protein sequence levels were 2. 5% and 0. 5%,respectively. Secondary structure prediction showed the changes in the number of alpha helices,extended strands,beta turns,and random coils. We thus reason that the mutation of the sigma factor may affect its binding to promoters,which leads to transcription of the PQQ gene and K. pneumoniae metabolic flux redistribution,and results in more efficient expression of PQQ.

关 键 词：肺炎克雷伯氏菌 Σ因子 RPOD 吡咯喹啉醌 

分 类 号：TQ464.8[化学工程—制药化工] Q789[生物学—分子生物学]