内生真菌Shiraia sp.Slf14竹红菌素生物合成途径及代谢调控

The hypocrellins are a unique class of perylenequinones characterized by a pentacyclic conjugated chromophore giving rise to photoactivity. The hypocrellins have been used for centuries in Chinese folk medicine for the treatment of vitiligo, psoriasis, and other diseases. There have been extensive studies of the hypocrellins and their derivatives for their use in photodynamic therapy for cancer treatment. Secondary metabolite biosynthesis mechanism has been the core issue of secondary metabolism studies, but so far perylenequinones biosynthetic pathways are few studied. Furthermore, hypocrellins biosynthesis mechanism is currently unknown. Shiraia sp. Slf14, which can produce hypocrellins, is a novel endophytic fungus isolated by our research group from Huperzia serrata in China, and the Shiraia sp. Slf14 genome sequence is first sequenced by our research team. Based on this, we first use bioinformatics methods to predict possible hypocrellins biosynthetic gene cluster. Secondly, we study hypocrellins biosynthesis key enzyme genes fuction according to analysis gene cluster transcriptional level and intermediate metabolite differences between wild type and mutant strain by gene knockout and covering technology. Lastly, we use quantitative RT-PCR technique to study environmental factors how to influence hypocrellins accumulation. This project will not only elucidate hypocrellins biosynthesis in Shiraia sp. Slf14, but also lay the foundation for heterologous production of perylenequinonoid pigment using metabolic engineering and synthetic biology technique in future.

竹红菌素是我国特有的一类苝醌类光敏色素，用于开发新型抗肿瘤抗病毒药物、光敏活性农药及新型光电转换材料等方面前景广阔。次级代谢产物生物合成机制一直是次级代谢研究的核心问题，但迄今有关苝醌类化合物生物合成途径的研究较少，而竹红菌素生物合成机制更是未见报道。本课题组前期获得产竹红菌素的内生真菌Shiraia sp. Slf14，并完成了其全基因组测序。本项目以此为基础，首先采用生物信息学方法预测可能的竹红菌素生物合成基因簇；再采用基因簇基因转录水平分析、基因敲除/回补技术对预测基因簇进行确证，通过分析野生型和基因敲除突变型菌株中间代谢产物的差异，对基因簇中关键基因进行功能鉴定；最后利用RT-PCR技术在转录水平上开展环境因子对竹红菌素代谢调控研究。本研究不仅能揭示竹红菌素的生物合成途径，为苝醌类色素生物合成机制研究提供资料，而且可为将来苝醌类色素的代谢工程、组合生物合成研究奠定基础。

苝醌类色素在开发新型抗肿瘤抗病毒药物、光敏活性农药及新型光电转换材料等方面前景广阔，但迄今有关苝醌类化合物生物合成途径的研究较少，而竹红菌素生物合成机制更是未见报道。本课题组开展了产苝醌类色素内生真菌Shiraia sp. Slf14全基因组测序，并预测了苝醌类色素生物合成基因簇，采用转录组测序技术对该基因簇进行了确证。在上述基础上，将预测的相关基因在酵母（S. cerevisiae BJ5464-NpgA）中进行了异源表达，成功实现了elsinochrome C(EC)的生物合成，验证了相关基因功能，明确了HYP1、2、3和4是EC合成的核心基因，并提出了苝醌类色素生物合成途径；发现果糖、酵母膏、精氨酸和丙苯氨酸以及金属离子Na+、Ca2+能促进苝醌类色素的合成，提出了Ca2+提升苝醌类色素生物合成的分子机制。本研究不仅揭示苝醌类色素的生物合成途径，而且为将来苝醌类色素的代谢工程、组合生物合成研究奠定坚实基础。