基于pacC信号途径研究pH调控红曲色素合成和分泌的分子机制

Monascus pigments (MPs), one of the main metabolites produced by Monascus spp., are a kind of natural colorants which have been widely applied in the food industy. Recently, researches have shown that pH can remarkably affect the composition and yield of MPs products. So, pH regulation is a potential low-cost technique for the MPs fermentation. However, the mechanism of the regulation of MPs biosynthesis and secretion by pH is all most unknown. In our previous study, based on the genomic database of M. ruber M-7, a putative pH signaling pathway,the pacC pathway, were discovered in Monascus spp..The pacC pathway is conserved in fungi and might be involved in the regulation of MPs biosynthesis and secretion. In the current proposal, the following studies will be carried out. Firstly, the effect of pH on MPs biosynthesis and secretion will be explored in further detail by analyzing the compositions, concentrations and structures of pigments both intra- and extra-cellular under different pH condition. Then, the key gene in the pacC pathway will be cloned and its related mutant strains will be constructed to investigate the association between the pacC pathway and MPs biosynthesis and secretion. Meanwhile, EMSA and DNase I footprinting experiments will be carried out to explore the relationships between the transcriptional factor pacC and regulator genes, synthase genes and transporter genes in the MPs gene cluster. These results in the study will provide theoretical and technological guidance to optimize and control of MPs fermentation process.

红曲色素(Monascus pigments，MPs)是红曲菌产生的一种天然食用色素，包括黄、橙、红三类色素组分，应用广泛。研究发现，pH能显著影响MPs发酵产物的组成和产量，是一种潜在的低成本的MPs发酵调控手段，其作用机制目前尚不清楚。本课题组从红色红曲菌M-7基因组中发现了可能参与响应pH信号并调控MPs基因簇转录的pacC信号途径。本项目拟在此基础上，分析不同pH条件下红曲菌M-7产生的胞内外MPs在种类、含量和结构方面的差异，系统研究pH对不同MPs组分合成和分泌的调控效应；构建pacC途径中关键基因的突变菌株，研究pacC途径在pH调控MPs合成和分泌过程中的作用；利用EMSA、DNase I足迹实验，分析转录因子pacC与MPs基因簇中调节基因、合成酶基因和运输蛋白基因转录调控的关系，以探讨pH影响MPs合成和分泌的分子机制，为MPs发酵过程的优化和控制提供理论与技术支撑。

红曲色素（Monascus pigments，MPs）是红曲菌（霉）（Monascus spp.）发酵产生的次生代谢产物，包括黄色、橙色和红色三类色素组份，是我国传统特色发酵产品，主要用于食品着色。研究发现，pH对红曲菌MPs的发酵生产有显著影响，是一种潜在的低成本的MPs发酵调控手段，但其作用机制尚不清楚，阻碍了相关技术的进一步开发与利用。本项目的研究内容主要包括：（1）利用液态缓冲培养体系，采用两步培养法，分析了红色红曲菌M-7在酸性条件（pH3、pH4、pH5和 pH6）、中性条件（pH7）和碱性条件（pH8）下胞内和胞外MPs组份在种类、含量和结构方面的变化规律，揭示了pH对MPs合成和分泌的调控效应；（2）克隆了红色红曲菌M-7 pacC信号途径中关键基因，即转录因子pacC，根据同源重组的原理构建了pacC基因敲除菌株和基因截短突变菌株，分析了两种突变菌株在不同pH培养条件下MPs合成和分泌的变化，发现pacC信号途径在pH调控MPs合成和分泌过程中发挥着重要作用；（3）结合MPs基因簇中各调节基因、合成酶基因和运输蛋白基因启动子区域的生物信息学分析结果，进行了转录因子与启动子的体外结合检测，发现聚酮合酶基因MrpigA、转录激活因子基因MrpigB和运输蛋白基因MrpigP启动子区存在pacC的结合位点，表明转录因子pacC与MPs基因簇间存在直接的转录调控关系。本项目的研究结果可用于指导MPs液态发酵过程的优化和高效控制，有利于促进红曲色素现代发酵技术的发展。