啤酒花聚酮化合物代谢途径系统解析及其合成生物学应用

Hop (Humulus lupulus L.), which belongs to Cannabaceae family, is an important ingredient, along with barley and yeast, for the beer brewing industry. Hops glandular trichomes uniquely produce two important classes of secondary metabolites in abundance: bitter acids and xanthohumol, which give beer its flavor and have been used as potential anticancer drugs and nutraceutical supplements. For these reasons, many studies had been performed on bitter acids and xanthohumol biosynthesis in the hop trichomes. However, several open questions about the enzymatic components and regulatory mechanism of the two biosynthesis pathways are still remained to elucidate. Recently we successfully functional identified the cytosolic CCLs (CoA ligase), together with mitochondrial thioesterases, which play a key role in linking BCAA degradation and bitter acid biosynthesis. We further proved that it was doable to engineer the bitter acids/xanthohumol pathways in yeast cell. The aim of this project is to continually increase our understanding of the components involved in bitter acids and xanthohumol pathway at molecular level by merging transcriptomics and metabolomics data and other modern techniques. ESTs will be mined for biosynthetic (related prenyltransferases and monooxygenase have top priority) and regulatory-related genes, and the functions of the interested genes will be elucidated by using molecular/biochemical techniques and hop transformation method. The metabolic engineered yeast strain will be constructed by using synthetic biology and "-omics" technologies for optimal production of bitter acids and xanthohumol. The knowledge generated from this project will lay a solid foundation for metabolic engineering of these high-value metabolites, as well as providing potential for hop molecular marker-assisted breeding.

啤酒花（Humulus lupulus L.属大麻科）和大麦、酵母一起，是啤酒酿造工业必不可或缺的原料。苦味酸和黄腐醇（在啤酒花腺体腺毛特异合成积累）作为啤酒风味物质基础，具有很高的药用/保健功效和经济价值。关于苦味酸和黄腐醇代谢途径的研究早已展开，但迄今对其代谢途径构成和调控机制仍不清楚。最近我们成功鉴定苦味酸代谢途径上游关键酶：CoA连接酶和硫酯水解酶，同时证明在酵母中将这两条途径进行代谢工程重组是可行的。本次项目中我们将在确定其它候选基因（利用转录组和代谢组数据关联分析）的基础上，综合应用分子生物学、生物化学和构建转基因啤酒花等手段系统解析参与黄腐醇/苦味酸代谢下游途径酶学构成（异戊烯基转移酶和单加氧酶等）和调控机制。我们将进一步利用合成生物学的技术在酵母中重组代谢途径生产苦味酸和黄腐醇，通过优化相关代谢模块获得高产工程菌株；项目产生的知识还可为培育风味品质上乘的啤酒花品种提供帮助。

本项目（2015.01-2018.12）计划通过对啤酒花腺体腺毛转录组和代谢组学的研究，在分子水平上解析苦味酸和黄腐醇生物合成途径，在克隆和功能验证相关结构基因的前提下，尝试将苦味酸和黄腐醇代谢途径在酵母中进行功能重组的可行性。通过四年的工作我们成功解析了苦味酸下游合成途径，证明以异戊烯基转移酶为锚定蛋白的膜蛋白复合体的存在；并将其在酵母体系中重组，证明了利用合成生物学策略重组整条代谢途径的可行性。同时发现两类没有催化功能的查尔酮异构酶分别通过蛋白-蛋白互作和蛋白-化合物互作提高黄腐醇的生物合成效率并稳定黄腐醇化合物的开环结构。相关结果发表高水平论文两篇（PNAS一篇，IF > 9.0; Plant Physiology一篇，IF > 6.0），获得专利3项，培养博士后一名和博士生两名，超额完成项目的各项既定目标。整个的项目经费都是按照年度预算计划进行。