灵芝中灵芝酸生物合成相关P450基因的鉴定与功能研究

Ganoderic acids (GAs), a kind of highly oxygenated lanostane-type triterpenoids, are important bioactive constituents of the famous medicinal mushroom Ganoderma lucidum. GAs are synthesized by mevalonate/isoprenoid pathway, and cytochrome P450 monooxygenases (P450s) are speculated to be involved in later steps of GA biosynthesis pathway.Identification and characterization of the related P450s are essential for understanding of GA biosynthesis and its regulatory mechanism. Recently, elucidation of the G. lucidum genome revealed that the cyp512 and cyp5144 families of P450s are likely to be involved in GA biosynthesis. In this project, an efficient gene targeting system will be first developed in G. lucidum using our previous established homologous genetic transformation system. Moreover, candidate cytochrome P450s gene will be disrupted and over-expressed in the genetically-engineered G. lucidum strain increased their homologous recombination frequency, in order to identify those cytochrome P450s involved in GA biosynthesis. Furthermore, the identified cytochrome P450s will be expressed in Saccharomyces cerevisiae to characterize their functions. This work will be helpful for comprehensive understanding of GA biosynthesis as well as for metabolic engineering of G. lucidum for GA production. Moreover, the related information will be helpful to the further research on secondary metabolism biosynthesis in other mushrooms, which are an important source of biopharmaceuticals and functional foods.

灵芝酸是灵芝中的重要活性成分，它是一类高度氧化的羊毛甾烷类衍生物。在灵芝酸的生物合成中，细胞色素P450酶参与了中间代谢产物羊毛甾醇的后修饰形成灵芝酸的过程。鉴定和研究相关的P450基因对于解析灵芝酸的生物合成及其代谢调控具有重要的意义。对灵芝的基因组序列进行分析，发现其中的CYP512和CYP5144家族的P450酶可能参与了灵芝酸的生物合成。本课题拟在我们前期建立的灵芝基因转化系统的基础上，构建同源重组效率提高的灵芝工程菌株。在工程菌株中通过缺失和过量表达CYP512和CYP5144家族的P450基因，分析基因缺失/过量表达对灵芝酸积累和灵芝酸生物合成相关代谢流量谱的影响，鉴定参与灵芝酸生物合成的P450基因。进一步在酵母中异源表达及功能验证关键的P450基因。本项研究有助于深入解析灵芝酸的生物合成及其调节机制，为今后广泛利用药用真菌生产有用次级代谢产物提供坚实的理论基础。

灵芝酸是灵芝中的重要活性成分，它是一类高度氧化的羊毛甾烷类衍生物。在灵芝酸的生物合成中，细胞色素P450 酶参与了中间代谢产物羊毛甾醇的后修饰形成灵芝酸的过程。鉴定和研究相关的P450 基因对于解析灵芝酸的生物合成及其代谢调控具有重要的意义。本研究首先建立了真菌灵芝的同源基因转化系统，并通过表达灵芝酸生物合成途径的关键基因验证了转化方法的有效性，提高了灵芝中抗癌物质灵芝酸的含量。首次在真菌灵芝中实现了异源基因的表达，提高了灵芝细胞中灵芝酸的积累。利用同源基因转化技术，缺失了灵芝的 Ku80 基因，并建立了灵芝的基因缺失/沉默平台。利用该方法成功抑制了灵芝中 Lig 4 和 Crz 等基因的表达。进一步通过 RNA-Seq 技术分析了可能参与灵芝酸生物合成的 P450 基因，克隆了14 个 P450 基因，通过基因表达量及灵芝酸积累的相关性分析，初步筛选了候选的 P450 基因。对目标 P450 基因进行基因沉默和过量表达获得了相应的工程菌株。通过分析野生型菌株、基因沉默菌株和基因过表达菌株中灵芝酸生物合成的情况，确定了CYP512V2基因参与了灵芝酸的生物合成。