红曲色素发酵产生过程中光磁耦合调控其生物合成的分子机理研究

Monascus pigments (MPs) as Chinese traditional natural food colorants are produced by Monascus spp.. In recent years, MPs are also the fastest growing food colorants in China. The low productive rate and high manufacturing cost of MPs are very urgent to be solved. In our previous studies we have found that the joint treatment to Monascus spp. with light and magnetic field, can significantly increase the MPs productive rate which is higher than being treated by separate light and magnetic field, and also found a magnetic receptor gene, a red light receptor gene and two green light receptor genes in Monascus genome but without any blue light receptor gene. In this project, Monascus ruber M7 and its light receptor gene deletion mutants will be taken as the starting research strains, and following researches will be done: Light-magnetism-joint treatment conditions to Strains M7 will be optimized; Transcriptomic and proteomic technologies will be applied to study the omics level variation of the strains, aiming to find possible blue light receptor gene(s) in Strain M7; Combining the protein co-purification with gene knockout technology, the relationship of the light and magnetic receptors in Strain M7, and the regulatory network and key gene(s) of their effects on the MPs synthesis will be investigated; Using markerless gene modification technology to construct a highly efficient and safe engineering strain which can be regulated by the joint treatment of light and magnetic field; The fermentation conditions of the engineering strain under the joint treatment of light and magnetic field will be optimized; The molecular mechanism that joint treatment of light and magnetic field increases MPs productive rate will be researched. The research results of this project will guide the construction of Monascus strains with high MPs yield in order to increase MPs yield with joint treatment of light and magnetic field.

红曲色素（Monascus pigments，MPs）是红曲菌又称红曲霉（M. spp.）发酵产生的天然食品着色剂，是我国传统产品，也是近年来我国增长速度最快的食品着色剂，但存在MPs产率低等问题。研究发现，光照和磁场耦合（联合）处理红曲菌时，能显著提高其MPs产率，且比光、磁单独处理时效果更显著，存在叠加作用；在红曲菌基因组中也发现了磁受体和红/绿光受体基因，但没发现目前已知的蓝光受体基因。本项目以红曲菌M7为研究对象，优化M7光磁耦合处理条件；研究M7在光磁耦合处理下组学水平变化，挖掘可能存在的新蓝光受体基因；以蛋白共纯化等技术，研究M7中光、磁受体相互作用，及其对MPs合成调控网络与关键基因；以基因无痕改造技术，构建光磁耦合调控MPs产生的高效安全菌株；优化其光磁耦合发酵条件；探讨光磁耦合提高MPs产生分子机理。研究结果将指导红曲菌高产MPs菌株构建，实现光磁耦合提高MPs发酵产率。

红曲色素（Monascus pigments，MPs）是由红曲菌，又称红曲霉（Monascus spp.）发酵产生的我国传统的天然食品着色剂，也是近年来我国增长速度最快的天然食品着色剂，但存在MPs产率低等问题。课题组前期研究发现，光照和磁场耦合处理红色红曲菌（M. ruber）M7等红曲菌时，能显著影响其MPs产率，且比光、磁单独处理时效果更显著，存在明显的叠加作用；在红色红曲菌M7基因组中也发现了磁受体（mr-magR）和红/绿光受体基因，但没发现目前已知的任何蓝光受体基因。本课题以红色红曲菌M7为研究对象，首先优化得到红色红曲菌M7的最佳蓝光和磁场耦合处理条件为“195 lx蓝光+10 mT磁场”，然后基于蓝光受体与磁受体的互作，以异源表达mr-magR获得的Mr-MagR蛋白为“诱饵”，成功在红曲菌M7的菌体蛋白中“钩钓”到新蓝光受体蛋白，并在其基因组中发现了新蓝光受体基因mr-blR1。在此基础上，为了同时对红曲菌M7中的多基因同时进行高效改造，构建了红曲菌M7的无痕高效基因改造体系Δmrlig4+mrpyrG::G，然后将蓝色荧光蛋白(BFP)和绿色荧光蛋白(BFP)基因分别与mr-magR和mr-blR1连接后，同时导入Δmrlig4+ mrpyrG::G中，成功获得了含双荧光蛋白基因菌株mr-magR-bfp-mr-blR1-gfp，激光共聚焦显微镜观察表明，Mr-MagR和Mr-BlR1在红曲菌M7中的确存在相互作用，同时表明mr-blR1为新蓝光受体基因。进一步以Δmrlig4+mrpyrG::G为出发菌株，构建了mr-blR1无痕改造菌株，并以组学分析技术发现，蓝光与磁场耦合（195 lx蓝光+10 mT磁场）调控MPs产生的机理是通过降低组蛋白H3K4的甲基化从而提高MPs的产生。研究结果对指导红曲菌高产MPs菌株的构建，实现光磁耦合提高MPs的产率奠定了基础，对光磁耦合调控其他微生物等细胞次级代谢的产生也有重要的指导意义。