产2S-甲基丙二酰辅酶A的基因工程假单胞菌的构建

Construction of an engineered strain of Pseudomonas producing 2S-methylmalonyl-CoA

目的假单胞菌是次级代谢产物生物异源表达的优良宿主菌,然而它缺少许多次级代谢产物生物合成所需的小分子前体2S-甲基丙二酸辅酶A(2S-mm-CoA),因此构建产生2S-mm-CoA的宿主菌将有利于次级代谢产物的异源表达。方法本研究将来源于大肠埃希菌的编码甲基丙二酰辅酶A变位酶的sbm基因和编码甲基丙二酰辅酶A变位酶复合物保护蛋白的argK基因以及来源于天蓝色链霉菌的编码甲基丙二酰辅酶A异构酶的epi基因通过同源重组整合到恶臭假单胞菌KT2440(Pseudomonas putida KT2440)基因组,得到基因工程菌株P.putida LS-MCM。结果气质联用(GC/MS)分析表明P.putida LS-MCM能产生达2.22nmol/mL的2S-mm-CoA。结论 P.putida LS-MCM可作为异源表达需要2S-mm-CoA作为前体单元的次级代谢产物的宿主菌。

Objective Pseudornonas is one good host for the heterologous expression of secondary metabolites, but it lacks 2S-methylmalonyl-CoA （2S-mm-CoA） that many secondary metabolites need as biosynthetic precursor, a 2S-mm-CoA producing Pseudomonas strain would be useful for the heterologous expression of secondary metabolites. Methods In this study, sbm gene coding mm-CoA mutase and argK gene coding mm-CoA mutase （both are of Escherichia coli origin）, and the Streptomyces coelicolor origin epi coding mm-CoA epimerase were integrated into the Pseudomonas putida KT2440 genome through homologous recombination, generating P putida LS-MCM. Results GC/MS analysis shows that the 2S-methylmalonyl-CoA yield is up to 2.22 nmol/mL. Conclusion P putida LS-MCM could be used as the host for the heterologous expression of the secondary metabolites required 2S- mm-CoA as precursor.