化学遗传学策略激活木生真菌桑黄抗肿瘤活性相关沉默次生代谢途径研究

Traditional medical fungi are un-fully exploited vital sources of new bioactive natural products. Most of fungal secondary metabolite pathways are silent under ordinary cultured conditions, which limit the biosynthetic effect of new bioactive products. The silent gene families in secondary metabolite pathways could be induced or activated by chemical genetic strategy, thereupon enriching the chemical molecules diversity of secondary metabolite. In our early experiments, Phellinus igniarius, wood-inhabiting fungal strain, was chosen and its EtOAc extract showed strong anti-tumor activity. The fungus produced abundant secondary metabolites with diverse molecular variety and anti-tumor activities when it was cultured in different media, suggesting that the metabolite biosynthetic pathway of P. igniarius could be selectively regulated or induced by many factors. Based on the previous study, in this project we are interested in the activation or induction of silent gene families related with anti-tumor metabolites in P. igniarius by adding molecules such as plant hormones (MeJA, methyl jasmonate) and enzyme inhibitor (suberoylanilide hydroxamic acid) et al., aiming to obtain novel anti-tumor active compounds from activated gene family expression products, and verify anti-tumor mechanism by molecular pharmacological methods, so as to providing latent leading compounds for anti-tumor drug research on Chinese traditional medicine. For the novel active compounds, how the small inductive molecules induce or regulate their biosynthesis are going to be further investigated, which might provide scientific basis for medical fungi uses and modernization of Chinese traditional medicine.

传统药用真菌是未被充分发掘的新颖活性天然产物的重要来源，但常规培养条件下真菌的很多次生代谢途径处于沉默状态，限制了活性新产物的生物合成效率；利用化学遗传学策略可诱导激活沉默基因簇，增加次生代谢产物分子化学多样性。前期研究发现，木生真菌桑黄（Phellinus igniarius）次生代谢产物具有优异抗肿瘤活性，且不同培养条件下的产物种类和活性各异，表明其沉默代谢途径可被选择性激活。据此，本项目拟利用酶抑制剂（SAHA）和植物激素（NAA，MeJA）等小分子激活桑黄次生代谢途径中抗肿瘤活性产物相关的沉默基因簇，从激活后的基因簇表达产物中分离获得结构新颖、抗肿瘤活性强的天然产物分子，通过分子药理学方法初步阐明活性机理，为中药抗肿瘤创新药物研究提供先导物；对活性好、结构新的化合物，进一步探讨小分子诱导物对活性产物生物合成途径的诱导和调控机理，为药用真菌资源发掘和中药现代化提供科学依据。

传统药用真菌中有多种天然生物活性成分尚未被发掘。桑黄（Phellinus sp.）在前期研究中表现出较好抗肿瘤活性，但有效成分未被鉴定。微生物中存在常规培养条件下处于沉默状态的次生代谢途径，利用化学遗传学策略可诱导激活沉默基因簇，增加次生代谢产物分子化学多样性。本项目对桑黄（Phellinus igniarius）开展了多种条件的培养、产物分析和活性测试，具体包括培养基（氮源、碳源和金属离子），培养时间，添加小分子等方面。（1）在不同条件下桑黄的次级代谢产物出现较大差异，筛选到4种代谢产物多样性增加的条件。这说明桑黄在常规条件下表达较弱或未表达的生物合成基因簇被某些特定的条件激活或加强。（2）从不同的培养产物中分离鉴定了10个类型各异的单体化合物，并进行了抗肿瘤和抗细菌的活性测试，发现具有较弱抗菌活性。（3）通过共孵育和二级质谱等方法，对活性天然产物姜黄素被小鼠肠道菌的生物转化进行了研究，发现姜黄素可以被肠道菌转化为抗氧化活性更强的成分，对转化后结构进行了推测鉴定。（4）通过异源宿主表达和体外生化反应，对真菌麦角生物碱类化合物的生物合成途径中进行了解析。本项目发表SCI论文2篇；结果为桑黄进一步开发利用提供了基础。