阿姆斯特丹散囊菌新化合物的发现及合成途径解析

Eurotium amstelodami, a member of core microbiota in traditional solid state fermentation, play important role in production of flavor components and quality of fermented foods, which have a large number of unknown silent biosynthetic gene clusters in genome. Novel bioactive compounds could be discovered using established methods to activate these silent gene clusters. In this study, we activate these silent gene clusters by microorganism co-culture and overexpression of LaeA, search the main variant phytochemistry and discovery of novel bioactive compounds by comparative analysis on HPLC fingerprints. Meanwhile, targeted variant compounds have been constructed and identified using chromatography and spectroscopy methods. The biosynthesis pathway of novel bioactive compounds were described using RNA-seq and real time PCR. These results would discover the relationship between silent gene cluster and novel compound, also illuminate the biosynthesis pathway of novel bioactive compounds and mechanism of regulation, and provide new insight into the safety of E. amstelodami, and promote the development of traditional food fermentation and health care product.

阿姆斯特丹散囊菌（Eurotium amstelodami）是中国传统发酵体系中广泛分布的优势菌种，对发酵产品风味前体物质和独特品质形成起着重要作用。基因组中存在大量功能未知的沉默生物合成基因簇，在特定条件下激活，有望产生多种新化合物。本项目拟采用共培养和超表达LaeA策略激活沉默基因簇，HPLC指纹图谱叠加比对，分析各样品化合物的差异及新化合物的产生，色谱学和波谱学技术进行纯化与结构鉴定；RNA-seq与定量PCR初步解析新化合物的生物合成途径。本课题实施，可明确沉默次生代谢生物合成基因与新化合物之间的关联性，解析生物合成途径及调控机制，进一步确证阿姆斯特丹散囊菌的安全性，指导传统发酵产业及相关保健医疗产品开发。

阿姆斯特丹散囊菌和冠突散囊菌是发酵茶、豆酱和酒醅等中国传统发酵体系中广泛分布的优势菌种，对发酵产品风味物质和独特品质形成起着重要作用，有巨大开发利用潜力。散囊菌属基因组中存在大量功能未知的沉默生物合成基因簇，多种策略激活产生可产生新化合物。本项目从传统发酵体系中系统分离各类散囊属真菌，蓝光照射、双氧水诱导，HPLC指纹图谱叠加比对分析，在代谢物图谱水平上对散囊属菌进行分类、鉴定；构建冠突散囊菌llm1基因（LaeA同源的甲基转移酶l）超表达菌株，以转录组和UPLC/Q-TOF-MS技术系统研究该基因过表达对冠突散囊菌形态发育，次级代谢物图谱的影响，解析新化合物产生机制。以阿姆斯特丹散囊菌和芽孢杆菌类、酵母类及其他属真菌共培养策略激活沉默基因簇，UPLC/Q-TOF-MS分析了阿姆斯特丹散囊菌和解淀粉芽孢杆菌、地衣芽孢杆菌共培养不同阶段的代谢物变化情况，转录组解析沉默基因激活机制及新化合物合成。以植物乳杆菌、地衣芽孢杆菌、酿酒酵母、冠突散囊菌及多种菌株混合发酵白参，系统分析白参发酵前后化学成分的变化及抗氧化与降血糖活性提升情况。在共培养和微生物互作研究基础上，构建合成菌群，融合中药材发酵，形成微生态制剂系列产品，已覆盖肉牛、生猪、蛋鸡、水产及宠物等领域，以微生态平衡理念解决生态养殖和医疗保健领域面临的瓶颈问题。