摘要
[Objective]To confirm the function of the farnesyl diphosphate(FPP)cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319.[Methods]orf2064 was expressed in Escherichia coli,and the recombinant protein was purified and assayed with FPP as the substrate.The reaction products were detected by GC-MS.An FPP-overproducing E.coli strain was engineered for heterologous expression of orf2064.The fermentation products were analyzed by GC-MS,and the target compound was isolated and structurally characterized by nuclear magnetic resonance spectroscopy(NMR).In addition,orf2064 was heterologously expressed in Streptomyces,and the fermentation products were analyzed by GC-MS.[Results]GC-MS revealed that both the in vitro reaction of the recombinant protein ORF2064 and the heterologous expression products in E.coli and Streptomyces consistently produced a compound with identical retention time and[M+]of m/z 204.Subsequent isolation,purification,and NMR analysis confirmed this compound as calarene.[Conclusion]The FPP cyclase encoded by orf2064 in S.exfoliatus is identified as an calarene synthase.
【目的】确认脱叶链霉菌UC5319中法尼烯焦磷酸(farnesyl diphosphate,FPP)环化酶基因orf2064的功能。【方法】采用大肠杆菌蛋白表达系统对orf2064进行表达,纯化重组蛋白,以FPP为底物开展体外反应,利用气相色谱质谱仪(GC-MS)检测产物。构建可高效合成FPP的大肠杆菌基因工程菌,对orf2064进行异源表达,通过GC-MS检测发酵产物,并从发酵产物中分离纯化目标产物,利用核磁共振(nuclear magnetic resonance spectroscopy,NMR)确定其结构。以链霉菌为宿主对orf2064进行异源表达,用GC-MS检测发酵产物。【结果】ORF2064重组蛋白体外反应产物、orf2064大肠杆菌异源表达产物和链霉菌异源表达产物在GC-MS分析中均显示产生了1种保留时间相同、分子量为204的产物,分离纯化后经NMR分析确认其为白菖烯。【结论】脱叶链霉菌中基因orf2064所编码的FPP环化酶为白菖烯合酶。
出处
《微生物学报》
北大核心
2025年第12期5294-5308,共15页
Acta Microbiologica Sinica
基金
国家重点研发计划(2021YFA0909500)
国家自然科学基金(81373305,31401057)。
