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

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%。

Abstract:

[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 Plt 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%.