基于代谢组学和转录组学的功能真菌C39发酵提高重楼药材甾体皂苷含量的生物转化机理研究

PARIDIS RHIZOMA is the main raw material of staple Chinese patent medicines such as Yunnan Baiyao and Gongxuening et al. Due to large market demand, little wild resources and slow growth under cultivation, its price is as high as 2000-3000 Yuan/kg. Steroidal saponins are the chief active ingredients of PARIDIS RHIZOMA, which has strong anti-tumor, hemostasis, and analgesic effects. In the early stage of our research, when endophyte C39 was fermented with the dried powder of PARIDIS RHIZOMA, the saponin content in it was greatly increased (the increase rates of different saponins reached 50%~130%), and its inhibitory effects on various tumors were significantly enhanced compared with its original raw medicinal materials. In order to study the biotransformation mechanism, liquid fermentation of endophytic C39 and the extract of PARIDIS RHIZOMA was set up. First, with the target compounds as indicator, the fermentation technique was optimized and established by multi-factor investigation, and verified by the anti-tumor activity of fermentation product. Then, the metabonomics method is used to analyze the overall metabolic profile of the chemical constituents during the transformation process, and the transcriptomics technique is used to study the changes of the fungal gene expression profile. The results of metabonomics and transcriptomics are integrated and analyzed. The biotransformation mechanism of endophytic C39 to increase the saponin contents of PARIDIS RHIZOMA can be revealed from two aspects of metabolic pathways and regulatory mechanisms. This research provides the experiment foundation for achieving the transformation of target polyphyllins by genetic engineering technique, improving the utilization of limited resources, and screening antitumor active compounds .

重楼是云南白药、宫血宁等大宗成药的主要原料，市场需求量大，但野生资源少，栽培生长缓慢，价格高达2000-3000元/公斤。重楼主要活性成分为甾体皂苷，具有较强的抗肿瘤、止血、镇痛等作用。课题组前期将滇重楼药材与功能真菌C39固体发酵，其皂苷含量大幅提高（各类皂苷的增长率达50%~130%），且对多种肿瘤的抑制作用较原药材显著增强。本研究拟以C39菌与重楼提取物液体发酵为模型，探讨其生物转化机理。首先以目标化合物为指标，通过多因素考察优化建立发酵工艺，并对发酵产物进行抗肿瘤活性验证。继而采用代谢组学方法分析转化过程中化学成分整体代谢轮廓，应用转录组学技术研究真菌基因表达谱的变化；将代谢组学和转录组学结果整合分析，从代谢途径和调控机制两个方面揭示C39菌生物转化提高重楼皂苷含量的作用机理。为实现以基因工程手段定向转化重楼皂苷目标成分，提高有限资源利用率，以及抗肿瘤活性化合物的筛选提供研究基础。

重楼是云南白药、宫血宁等大宗成药的主要原料，市场需求量大，但野生资源少，栽培生长缓慢，药材价格较高。重楼主要活性成分为甾体皂苷，具有较强的抗肿瘤、止血、镇痛等作用。课题组前期将滇重楼药材与功能真菌C39固体发酵，其皂苷含量大幅提高，且对多种肿瘤的抑制作用较原药材显著增强。本研究采用液体发酵的方法探讨C39菌提高重楼皂苷含量和抗肿瘤活性的机理，首先以重楼皂苷I、Ⅱ、Ⅶ、偏诺皂苷元以及薯蓣皂苷元为指标确定了C39菌与重楼提取物液体发酵的最佳工艺，抗肿瘤活性验证表明发酵产物对肝癌、宫颈癌和肺癌细胞有更强的抑制作用。通过对发酵前后的化合物代谢轮廓分析，筛选出了36个具有显著变化的差异代谢物，包括呋甾型皂苷、螺甾型皂苷、纽替型皂苷和苷元，根据各类皂苷成分的动态变化趋势推测发酵过程中发生了呋甾皂苷向纽替皂苷或螺甾皂苷的转化、糖基水解以及羟基或羰基的取代。在开展C39菌基因组测序的基础上，通过转录组对发酵过程中真菌基因表达谱进行分析，筛选出53个涉及甾体皂苷合成及转化的差异基因，发现C39菌依赖MVA途径进行甾醇合成，在糖苷酶、糖基转移酶、氧化还原酶和甾醇合成相关基因的作用下，实现了薯蓣皂苷元的合成，改变了甾体皂苷的构型、糖链和取代基，从而促进了甾体皂苷成分的转化。代谢组学和转录组学研究结果从代谢途径和调控机制两个方面揭示了C39菌生物转化提高重楼皂苷含量的作用机理，为实现以基因工程手段定向转化重楼皂苷目标成分，提高有限资源利用率，以及抗肿瘤活性化合物的筛选提供研究基础。