类胡萝卜素是一类具有抗肿瘤、抗炎等多种功效的天然色素，常用的类胡萝卜素是脂溶性的，生物利用度差，极大地限制了其在医药、食品等上的应用。蛹虫草类胡萝卜素含有脂溶性和水溶性两种，其中水溶性种类是迄今发现的生物利用度最好的类胡萝卜素，这预示蛹虫草类胡萝卜素具有特殊的生物合成途径。本项目拟采用SMURF和antiSMASH等工具结合比较基因组方法，发掘蛹虫草基因组中类胡萝卜素生物合成途径的关键酶基因；通过基因克隆、体外表达和底物饲喂试验鉴定基因功能；同时采用CRISPR等基因组编辑技术定点敲除目标基因，检测分析突变株缺失峰和/或增加峰的产物结构等代谢组学性质，解析突变株的类胡萝卜素及其中间产物的合成特性，确证敲除基因在蛹虫草类胡萝卜素生物合成中的功能及其调控作用。本项目开创蛹虫草类胡萝卜素生物合成等代谢组学研究的先河，为食药用菌代谢组学研究提供典型范例，具有重要科学意义和应用价值。

Carotenoids are a class of natural pigments with anti-tumor and anti-inflammatory properties. Common carotenoids are fat soluble with poor bioavailability, which largely restricts their applications in pharmaceuticals and food etc. Carotenoids of Cordyceps militaris contain both fat soluble and water soluble carotenoids, in which water soluble one is the best carotenoid with the highest bioavailability found so far. This implies that carotenoids of Cordyceps militaris have their special biosynthesis pathway. This project will firstly explore genes in the genome of Cordyceps militaris encoding enzymes for biosynthesis of carotenoids through bioinformatics tools of SMURF and antiSMASH with combination of comparative genomics methods. Then the functions of the genes will be identified and characterized by gene cloning, in vitro expression and substrate feeding experiments. The putative target genes involved in carotenoid synthesis will be site directed knocked out/deleted by CRISPR genome editing technology. Product structures of deletion peaks and/or additional peaks derived from knockout mutants will be determined and analyzed through metabonomic methods. The biosynthesis characteristics of carotenoids and their intermediate products of knockout mutants will be elucidated through metabonomic and genomic analyses. The functions and their expression regulations of the knockout genes will be further characterized and confirmed through these analyses. This project not only creates a precedent for the study on the metabonomics of carotenoid synthesis of Cordyceps militaris, but also provides a typical example for the study on the metabonomics of edible and medicinal fungi, showing that this has important scientific significance and practical application value.