假单胞菌M18中藤黄绿菌素合成限速酶基因的鉴定及表达优化  

Identification and expression optimization of the genes encoding rate-limiting enzymes in pyoluteorin biosynthesis of Pseudomonas sp.M18

作　　者：李赛男[1] 李慷[1] 王姝芸[1] 王国昊[1] 黄显清[1] 许煜泉[1] 

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240

出　　处：《微生物学通报》2012年第3期300-308,共9页Microbiology China

基　　金：国家自然科学基金项目(No.30800009);国家863计划项目(No.2007AA02Z215);国家973计划项目(No.2009CB118906)

摘　　要：【目的】假单胞菌株M18中负责抗真菌剂藤黄绿菌素(Plt)合成的结构基因包括pltLABCDEFG、pltM。为了鉴定Plt合成限速酶的基因,分别将9个结构基因过表达。【方法】以M18菌株染色体DNA为模板,PCR扩增这9个Plt合成基因的编码区,分别克隆到穿梭载体pME6032的tac启动子下游,构建9个结构基因的过表达载体,并转入假单胞菌株M18中。在KMB培养基中进行Plt发酵分析。【结果】分别携带pltC、pltD、pltF过表达载体的菌株与携带空质粒的菌株相比,Plt产量分别提高了96%、78%、75%。对重组菌株进行IPTG诱导浓度和诱导时间的优化,确定IPTG最佳诱导浓度为1.0 mmol/L,最佳诱导时间为培养6 h。【结论】pltC、pltD、pltF分别编码的Ⅰ型聚酮合成酶、卤化酶、乙酰CoA合成酶可能为Plt生物合成的限速酶。按照优化条件发酵,携带pltD、pltF过表达质粒的菌株产量分别上升77.5%、159.1%。[Objective] In Pseudomonas sp. M18, there are nine structural genes encoding pyoteorin （Plt） biosynthase, including pltLABCDEFG and pltM. In order to identify the gene incoding rate-limiting enzyme, 9 structural genes were overexpressed. [Methods] The entire ORFs of these nine genes were PCR amplified from the M18 strain chromosomal DNA template and respectively cloned into the Pseudomonas-E. coli plasmid pME6032, generating nine overexpression plasmids of plt genes. The M18 strains, carrying these overexpression plasmids, were assayed for Plt production in KMB media. [Results] It was showed that the Pit production of three strains respectively harboring pltC, pltD, pltF overexpression plasmids was raised by 96%, 78%, 75% as compared to the control. The optimal inducing concentration of IPTG was 1.0 mmol/L, and the optimal inducing time was at 6 hours after inoculation. [Conclusion] The genes pltC, pltD, pltF divergently encode polyketidesynthase, halogenase and acyl-CoA＂ synthetase rate-limiting enzyme in Plt biosynthesis. At the optimal IPTG concentration and inducing time, pyoluteorin production of strains containing pltD, pltF overexpression plasmids was respectively increased by 77.5% and 159.1%.

关 键 词：假单胞菌株 藤黄绿菌素 限速酶 IPTG诱导条件优化 

分 类 号：Q933[生物学—微生物学]