东乡野生稻内生链霉菌PRh3中Beta-咔啉生物碱Kitasetaline/Novkitasetaline的发掘与新颖生物合成机制的研究

东乡野生稻内生菌Streptomyces sp. PRh3激活后能够生产含S-乙基-N-乙酰半胱氨酸特殊结构的Beta-咔啉生物碱Kitasetaline及其具有良好抗疟原虫活性的新色胺衍生物Novkitasetaline，相关生物合成途径具有显著的菌株特异性，其所含有的新颖咔啉骨架合成和后修饰机制及催化酶极具研究价值。鉴于此，本项目拟通过开展：1）生物合成基因（簇）的体内失活和反式回补鉴定与异源表达分析；2）构建的各基因工程菌株中代谢中间体和结构衍生物的分离鉴定及活性优选；3）骨架合成Pictet-Spenglerase（PS酶）、含C-S键侧链组装催化酶及酰胺键合成酶等关键酶的发现与酶学机理研究等工作，阐明Kitasetaline/Novkitasetaline独特的生物合成机制，发掘具有自主知识产权的药用先导化合物与生物催化剂，促进我国特有的东乡野生稻内生放线菌资源的综合利用。

Kitasetaline and its new antiplasmodial tryptamine derivative Novkitasetaline are two beta-carboline alkaloids containing rare S-ethylated N-acetylcysteine moiety. These two compounds, isolated from the activated endophytic Streptomyces sp. PRh3 derived from Dongxiang wild rice, are biosynthesized through strain-specific beta-carboline backbone assemble line and post-modification pathway. To study this distinct mechanism of Kitasetaline/Novkitasetaline biosynthesis, we plan i) to identify the essential biosynthetic genes in vivo, by gene inactivation, gene trans complementation and heterologous expression of gene (cluster); ii) to isolate and identify intermediates and derivatives from generated mutant strains, combined with bioactivity evaluation of these obtained molecules; iii) to biochemically characterize the Pictet-Spenglerase responsible for beta-carboline skeleton formation and other enzymes involved in S-ethylated N-acetylcysteine moiety (C-S bond) formation and uploading tryptamine. In addition, our innovative research could generate new drug leads and biocatalysts with independent intellectual property rights. This will facilitate the utilization of endophytic actinomycetes as invaluable resources from Dongxiang wild rice.