三种不同磷酸烯醇式丙酮酸羧化酶基因的克隆、表达及其对宿主菌生长的影响

Cloning and expression of three distinct phosphoenolpyruvate carboxylases in Escherichia coli and their influences on cell growth

应用PCR技术分别克隆了集胞藻6803、鲍曼不动杆菌和大肠杆菌的磷酸烯醇式丙酮酸羧化酶(PEPC)基因,构建重组大肠杆菌。SDS-PAGE凝胶电泳结果显示,来自集胞藻6803和大肠杆菌的PEPC实现了高效表达,而来自鲍曼不动杆菌的PEPC表达较弱,提示密码子偏好性的影响。前两菌提前进入对数生长期,来自鲍曼不动杆菌PEPC工程菌却延迟生长,但这3种重组菌发酵24 h后的生物量与对照菌几乎相同。如果排除质粒复制造成的代谢负荷,过表达PEPC促进了宿主菌的生长,推测是因为重排了代谢流量。

We have engineered three recombinant E.coli overexpressing distinct phosphoenolpyruvate carboxylase(PEPC) from Synechocystis sp.PCC 6803,Acinetobacter baumannii and Escherichia coli.SDS-PAGE analysis showed high expression of PEPC except in the case of the PEPC from A.baumannii.Consistent with this result,unlike the E.coli harboring PEPC from A.baumannii,the other two recombinants overexpressing PEPC entered a log phase earlier than a control that carried a blank vector.More interestingly,all above three recombinant E.coli strains presented nearly equal biomass to wild-type strain after 24 h fermentation.Taken together,if excluding the metabolic burden arising from plasmid replication,PEPC could be a general driving force facilitating cell proliferation due to the reallocation of metabolic flux.