深海链霉菌来源的四种双吲哚生物碱中骨架合成基因的鉴定及功能研究

Spiroindimicins and indimicins, belonging to bisindole alkaloids group and displaying cytotoxic effects on some tumor cell lines, were isolated from the deep sea-derived Streptomyces sp. SCSIO 03032. It is noteworthy that the structures of these compounds are distinct from the known bacterial bisindole alkaloids. Spiroindimicin A possesses an unusual [5,6] spiro-ring skeleton, and spiroindimicins B - D contain a distinct [5,5] spiro-ring skeleton, while indimicins A - E bear a unique 1′,3′-dimethyl-2′-hydroindole. These new structures imply that there may present intriguing machinery in their biosynthetic pathway. In previous study, we have identified a partial gene cluster (spm) responsible for the biosynthesis of spiroindimicins and indimicins, and proposed the key genes for the formation of the core skeletons should be located somewhere of the genome. In this project, we aim to identify and characterize those dissociative genes. As a consequence, the project will focus on the following aspects: 1) identification of the genes for the formation of spiro-rings in spiroindimicins, and figuring out their biosynthetic pathway; 2) characterization of the key enzymes and elucidating the mechanism for the biosynthesis of 1′,3′-dimethyl-2′-hydroindole in indimicins; 3) expanding the structural diversity of bisindole alkaloids via combinatorial biosynthesis.

Spiroindimicins和indimicins是从深海链霉菌Streptomyces sp. SCSIO 03032中分离鉴定的双吲哚生物碱，具有较强的细胞毒活性。其中spiroindimicins骨架中含有新颖的5-5螺环或5-6螺环，indimicins骨架中包含两种去芳构化的二甲基吲哚，这与已知的细菌来源的双吲哚生物碱明显不同，从而提示其生物合成过程中蕴含独特的酶学机制。前期研究中我们克隆鉴定了参与spiroindimicins和indimicins生物合成的基因簇（spm）并推测催化其骨架合成的关键基因游离在spm基因簇外。在此基础上，本项目拟开展以下研究：1）定位分散在基因组中催化5-5螺环和5-6螺环形成的游离基因，解析两种螺环的形成机制；2）鉴定二甲基吲哚合成酶并阐明吲哚环去芳构化的酶学机理；3）利用组合生物合成技术获得更多结构新颖的双吲哚生物碱。

Spiroindimicins和indimicins是从深海链霉菌Streptomyces sp. SCSIO 03032中分离鉴定的双吲哚生物碱，对肿瘤细胞具有较强的细胞毒活性。其中spiroindimicins骨架中含有新颖的5-5螺环或5-6螺环，indimicins骨架中包含两种去芳构化的二甲基吲哚，提示其生物合成过程中蕴含独特的酶学机制。本项目在前期克隆鉴定spiroindimicins和indimicins主生物合成基因簇（spm）的基础上对游离在spm基因簇外负责其骨架合成的基因展开研究，取得以下研究成果：（1）通过生物信息学分析、体内基因缺失和生物转化实验鉴定了游离在spm基因簇外负责催化5-5螺环和5-6螺环形成的细胞色素P450酶ISGA0619，基于蛋白结构预测和底物对接结果解析了两种螺环的形成机制；（2）通过生物信息学分析、体内基因缺失鉴定了游离在spm基因簇外负责合成二甲基吲哚的甲基转移酶ISGA5680，利用化学合成中间体和生物转化实验证实了indimicins骨架的形成过程；（3）利用合成生物学技术构建spiroindimicins和indimicins生物合成基因缺失突变株，分离鉴定了11个新颖的结构类似物，推导了spiroindimicins和indimicins的生物合成途径。本项目为利用组合生物合成技术对双吲哚生物碱进行结构改造奠定了基础，同时为利用基因组挖掘获取含螺环或二甲基吲哚结构的新颖生物碱提供了探针基因，此外本项目还为其他生物合成基因非成簇排列的化合物的生物合成研究提供了可行性策略。