棒柄杯伞中抗肿瘤杂萜clavilactones的生物合成机制研究

Clavilactones是高等真菌棒柄杯伞产生的一类具有独特的苯并十元环结构单元的杂萜类化合物，由于优异的酪氨酸激酶抑制活性被认为是一类潜在的抗肿瘤先导化合物。从结构上看clavilactones来源于香叶基对苯二酚，但其生物合成特别是形成苯并十元环结构单元的生化和遗传基础完全不清楚。前期研究中通过代谢产物谱监测，同位素标记的前体饲喂，全基因组测序，转录组测序和定量PCR实验我们初步确定了一个可能负责clavilactones生物合成的基因簇。本项目拟在此基础上利用多种异源表达系统（大肠杆菌，酿酒酵母，构巢曲霉）对候选基因簇中关键基因进行异源表达并通过底物饲喂及体外酶催化实验对其功能进行逐一验证，进而完成clavilactones生物合成途径的重构。期望在基因和酶学水平上阐明其生物合成机制，发现催化机制新颖的构建苯并十元环的环化酶，为利用合成生物学制备和改造该类化合物奠定基础。

Clavilactones, a class of meroterpenoids with a ten-membered carbon ring fused to an aromatic ring produced by higher fungi Clitocybe clavipes, are potential antitumor lead compounds for their highly effective inhibition of tyrosine kinase. Although clavilactones seems to be biosynthesized from geranylhydroquinone, the biochemical and genetic basis of its biosynthesis has remain obscure. In previous study, we has identified a candidate gene cluster by metabolite profiling, isotope-labeling precursor feeding, genome sequencing, transcriptome sequencing and qRT-PCR. In this project, the functions of key enzymes involved in the biosynthesis of clavilactones will be characterized by heterologeous gene expression (E.coli, S. cerevisiae and A. nidulans), substrates feeding and in vitro enzymatic assays. The biosynthetic pathway of clavilactones will be reconstructed in A. nidulans. Deciphering clavilactones biosynthesis will reveal unique cyclase responsible for the biosynthesis of ten-membered carbon ring fused to an aromatic ring and lay the foundation to produce clavilactones or to engineer its pathway by using synthetic biology approaches.