红曲色素和桔霉素代谢分支途径的关键酶系及功能分析

Mycotoxin citrinin is an important food safety problem for Hong Qu (HQ) products with high color value. Both Monascus pigments (MPs) and citrinin (CIT) were secondary metabolites biosynthesized by the polyketide pathway in filamentous fungi Monascus, which their formation at initial phase were similar and then biosynthesized by different branching pathways. Therefore, there was a positive correlation of secondary metabolite biosynthesis between MPs and CIT. However, studies on the regulation of key enzymes on branching pathway of secondary metabolites biosynthesis of MPs and CIT, to achieve MPs increment and CIT reduction have been rare investigated yet. Representative of Monascus strains (high MPs with high CIT, high MPs with low CIT, and low MPs with high CIT), as researching object in this project, were selected from the germplasm appraising and resource bank of Monascus with special industrial production value built by our laboratory based on the previous researches. Based on the metabolic kinetics of MPs and CIT, targeting differential proteins expression at peak periods of secondary metabolite biosynthesis by using stable isotope labeled quantitative proteomics methods of both DIA (Data Independent Acquisition Analysis) and PRM (Parallel Reaction Monitoring Techniques) with RNA sequencing to verify each other, in order to qualitative and quantitative analyze the differentially expressed proteins (DEPs) and the differentially expressed genes (DEGs) in the polyketide biosynthetic pathway with their biological functions. Finally, the key enzymes with biological functions at the branch of biosynthetic pathway of MPs and CIT would be accurately identify from protein level and RNA level simultaneously, for providing the innovative thought on the controllability of industrial HQ production with high MPs and less CIT.

真菌毒素桔霉素是高色价红曲制品的重要食安问题。红曲色素（MPs）和桔霉素（CIT）均由真菌红曲霉通过聚酮体途径合成，起始阶段相似，之后产生不同的代谢分支，二者存在伴生代谢正相关性关系。通过调控MPs和CIT合成分支途径的关键酶系，实现高产色价低产桔霉素的研究尚属空白。本项目拟以本实验室特有工业化生产价值的红曲霉菌专用种质资源库中筛选的代表性高色高毒、高色低毒和低色高毒菌株为对象，基于MPs和CIT的代谢动力学研究基础，以代谢峰值期差异蛋白为靶标，利用基于稳定同位素标记（DIA）和平行反应监测（PRM）的定量蛋白质组学技术，辅以转录组测序技术（RNA-seq）进行互相验证，对聚酮体途径各通路差异蛋白和差异基因进行定性、定量、功能以及参与代谢途径等相关生物学分析，从蛋白水平和RNA水平同时精准解析MPs和CIT合成分支途径的关键酶系及其功能，为高色价红曲制品的桔霉素可控化生产提供创新思路。

真菌毒素桔霉素是红曲制品的重要食安问题。红曲色素（MPs）和桔霉素（CIT）均由真菌红曲霉通过聚酮体途径合成，呈伴生代谢正相关性关系，过程涉及大批基因的表达及网络式的调控，但目前通过分析和调控MPs和CIT的关键酶系，实现高产MPs低产CIT的研究尚属空白。本项目以筛选的代表性紫色红曲霉（高产MPs低产CIT菌株M34、高产MPs高产CIT菌株M3和低产MPs低产CIT菌株M69）为研究对象，系统研究了各代表性菌株对生长和代谢MPs和CIT的影响规律；采用比较基因组学方法，结合系统基因组学UFCG分析，从基因层面评估了代表性菌株种内具有较高的遗传相似性；利用RNA-seq和DIA技术对代表性菌株代谢峰值期的转录组和蛋白质组进行了系统分析，在M34 vs M3、M3 vs M69、M34 vs M69三组样本中，分别鉴定出964、2893、2558个差异表达基因和365、1256、1210个差异表达蛋白；通过GO功能和KEGG通路富集分析了差异基因和差异蛋白分布和可能行使的生物学功能、参与的代谢途径或信号通路，发现转录组与蛋白质组分析部分结果存在差别。因此，通过高通量多组学数据的关联分析，共鉴定出98、522、474个差异蛋白可找到相应的基因表达信息，其中82、452、429个与差异基因表达趋势相同的差异蛋白，主要涉及线粒体膜、催化活性、氧化还原活性、小分子物质和脂质的生物合成和代谢过程等方面的功能，主要参与了脂肪酸生物合成及代谢、氨基酸（丙氨酸、天冬氨酸、谷氨酸、烟酸盐、烟酰胺等）、丁酸和生物素代谢等通路；通过关联聚类分析发现，部分差异蛋白与其相关的转录本表达模式相反的，推测差异基因蛋白表达水平调控很可能与转录后调控有关。另外，关联分析发现pksCT、citD、mrl5、citE、citC及其编码的蛋白CIT聚酮合酶CitS、脱氢酶CitD、乙醛酸酶型结构域MRL5、脱氢酶CitE、氧化还原酶CitC是限制CIT生物合成的关键蛋白（基因）。通过全景式的多组学整合关联分析，实现其间的互补与整合，为阐明MPs和CIT的代谢调控机制提供了科学依据，为高色价红曲制品的CIT可控化生产提供创新思路，具有较高的学术价值和实践意义。