深海放线菌Streptomyces sp. SCSIO 03032抗肿瘤天然产物Spiroindimicins生物合成研究

Spiroindimicins A-D, four new natural products of indolocarbazole family, were isolated from deep sea actinomycetes Streptomyces sp. SCSIO 03032 in our research group. The structures of these compounds contain rare spiro unit. All compounds except spiroindimicin A exhibited in vitro selective cytotoxicities against several human cancer cell lines HepG2, CCRF-CEM, NCI-H460, and MCF-7 and the mouse melanoma cell line B16. As far as we know, chemical synthesis or biosynthesis of spiroindimicins has not been reported. In this project, whole genome of Streptomyces sp. SCSIO 03032 is sequenced and its genomic library is also constructed. The whole sequence of predicted spiroindimicins gene cluster is obtained with the aid of bioinformatics. Gene knockout and complementation analysis is used to indentify in vivo function of genes involved in spiroindimicins biosynthesis. Heterologous expression of the genes is used to elucidate novel catalysis mechanism of key enzymes that are responsible for biosynthesis of some important spiro unit. Combinatorial biosynthesis of spiroindimicins provides a new blueprint for rational generation of some new spiroindimicins derivatives as lead drugs with improved antitumor effect.

Spiroindimicins是本课题组从一株深海链霉菌Streptomyces sp. SCSIO 03032中分离的新天然产物，含有独特的螺环结构。spiroindimicins B-D对测定的肿瘤细胞显示良好选择性细胞毒性，对于抗肿瘤药物研发具有重要意义。至今spiroindimicins生物合成研究尚未开展。本项目拟对Streptomyces sp. SCSIO 03032全基因组测序及构建全基因组文库，结合生物信息学分析，克隆完整的spiroindimicins生物合成基因簇；利用敲除和回补技术，鉴定spiroindimicins生物合成基因的体内功能；利用体外异源过表达技术，挖掘负责螺环等特殊结构单元生物合成的新颖功能酶；阐明spiroindimicins的生物合成机制；探索组合生物合成，为理性创造基于spiroindimicins的新型高效抗肿瘤先导化合物，提供新策略。

Spiroindimicins是由深海来源的链霉菌Streptomyces sp. SCSIO 03032双吲哚生物碱类天然产物。结构上，Spiroindimicins具有独特的螺环结构。活性上，spiroindimicins B和C具有良好的抗肿瘤活性。本项目完成了Streptomyces sp. SCSIO 03032全基因组测序、全基因组文库的构建及spiroindimicins骨架生物合成基因簇的克隆。生物信息学分析表明，该主体基因簇包含3个吲哚咔唑骨架合成基因，2个色氨酸卤代修饰基因，1个黄素依赖单加氧酶基因，1个转录正调控基因及1个转运蛋白基因。对克隆的Spiroindimicins骨架生物合成基因簇于Streptomyces lividans TK64异源表达，发现负责螺环合成的关键基因游离于主体基因簇之外。已对spiroindimicins生物合成基因簇8个关键基因进行敲除，从突变株中分离得到5个新结构spiroindimicins生物合成中间体或衍生物。探索了5个关键酶的体外生化功能。通过基因敲除及前体喂养，已证实SpmNMT-1蛋白催化的甲基化功能为spiroindimicins螺环形成的必要步骤。确定了lynamicin D是Spiroindimicin A和D 的前体化合物，而lynamicin A是Spiroindimicin B和C的前体化合物。该研究推进了Spiroindimicins生物合成酶学机制的揭示，有助于利用代谢工程及组合生物合成创制新结构双吲哚生物碱类化合物。已发表SCI论文1篇，另1篇SCI论文正在整理。