蛹虫草类胡萝卜素生物合成途径解析及重要基因功能研究

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.

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。

蛹虫草是一种著名的食药两用真菌，含有亲水性更强的新型类胡萝卜素，本项目对其生物合成途径进行了系统研究，获得了以下结果：.（1）从蛹虫草类胡萝卜素的转录组测序获得了不同培养基的样品的差异基因936个，不同光照培养的样品1722个。挖掘出4个可能与蛹虫草黄色素合成相关的基因（CmTns、CmFhp、CmMox、CmHyp）。.（2）建立了蛹虫草的基因敲除系统，首次把split-marker方法成功应用于蛹虫草，通过构建CmFhp、CmMox、CmHyp基因的敲除载体（pCAMBIA0390-Bar-KOFhp、pCAMBIA0390-Bar-KOMox、pCAMBIA0390-Bar-KOHyp）和制备split-marker敲除载体，成功敲除了CmFhp、CmMox、CmHyp基因，并获得了敲除株ΔCmFhp、ΔCmMox、ΔCmHyp。.（3）建立了蛹虫草的基因回补系统，构建了CmFhp、CmMox、CmHyp基因的回补载体（pCAMBIA0390-Ben-ComFhp、pCAMBIA0390-Ben-ComMox、pCAMBIA0390-Ben-ComHyp），通过农杆菌介导转化法成功把CmFhp、CmMox、CmHyp基因的表达盒分别回补入了ΔCmFhp、ΔCmMox、ΔCmHyp菌株的基因组，获得了回补菌株ComFhp、ComMox和ComHyp。.（4）从蛹虫草基因组中克隆了蛹虫草类胡萝卜素合成的相关基因八氢番茄红素合成酶基因（Psy），圆酵母素加氧酶基因carT和醛脱氢酶carD基因。.（5）从蛹虫草类胡萝卜素中分离鉴定出一个新组分CMPP-2的分子结构，并将其命名为虫草烯（cordycepene）。虫草烯在278、449和477 nm处检测到最大吸收波长，分子量大小为263.1158 m/z，分子式为C14H17N1O4，是一种具有全新结构的新型色素。