红曲霉萃取发酵中非离子表面活性剂诱导的新黄色素生物合成途径解析

Extractive fermentation with nonionic surfactants can relieve the feedback inhibition of intracellular Monascus pigments to increase the production of Monascus pigments. In the previous study, we found that, nonionic surfactants can not only export intracellular pigments to the extracellular broth, but also induce to form four new yellow pigments that accounted for a large proportion of the extracellular pigments. At present, many researches have focused the rule and mechanism of the exportation of intracellular pigments to the extracellular broth in extractive fermentation, however, the biosynthetic pathway of the new yellow pigments remains unclear. In this study, first, the four new yellow pigments will be isolated and their chemical structures will be identified. Combining with their structural characteristics, their precursors will be verified by feeding experiment with isotope-labelled precursors and their biosynthetic pathway will be supposed. Through the analysis of RNA-Seq and whole genome information, the changes of global gene expression level will be analyzed, and the possible key biosynthetic genes of the new yellow pigments will be mined. The function of the key genes in pigment biosynthesis will be verified by gene knockout, heterologous expression and in vitro catalysis. Our aim is to elucidate the biosynthetic pathway of the new yellow pigments induced by nonionic surfactants. The results can establish theoretical basis for the targeted regulation and productivity increase of the industrial production of the new yellow pigments.

添加非离子表面活性剂的萃取发酵可以减轻胞内红曲色素反馈抑制并提高其产量，显示了其在红曲色素生产应用上的潜力。申请人前期研究发现，非离子表面活性剂不仅可以将胞内红曲色素萃取至胞外，还诱导胞外大量积累四种新黄色素组分。目前，对于萃取发酵中胞内红曲色素向胞外运输的规律及机制已有大量的研究报道，但对非离子表面活性剂诱导的新黄色素生物合成途径还未明晰。本项目首先对萃取发酵的新黄色素进行分离纯化和化学结构鉴定，结合其化学结构，采用稳定同位素标记前体饲喂实验确定其合成前体并推测其合成途径。结合RNA-Seq及红曲霉全基因组信息分析非离子表面活性剂诱导下的全局基因表达水平变化并挖掘新黄色素合成的关键基因，采用基因敲除以及异源表达体外催化实验，分析验证关键基因在新黄色素合成途径中的具体作用，解析非离子表面活性剂诱导的新黄色素生物合成途径，为其发酵生产上的定向调控和产率的进一步提高奠定理论基础。

添加了非离子表面活性剂的红曲霉萃取发酵可以将胞内脂溶性色素萃取至胞外，缓解胞内产物抑制，增加色素产量，同时还可以在胞外大量产生新的黄色素组分，目前对于萃取发酵形成新黄色素的机制尚未明晰。因此，本研究系统地对比了萃取发酵和常规发酵在细胞生理和代谢上的差异，发现萃取发酵极大地影响了细胞膜脂肪酸组成、胞内油脂含量及细胞表面微观状态，萃取发酵胞外黄色素的生成和胞内NADH/NAD+值正相关；采用转录组和蛋白组技术对萃取发酵不同时期全局转录调控及蛋白表达调控进行分析，结合转录-蛋白组关联分析、GO及KEGG富集分析，构建了萃取发酵全局代谢调控网络，萃取发酵通过上调糖酵解通路及转录因子ctnF表达为细胞色素、脂类物质合成提供充足小分子前体，通过上调磷酸戊糖途径、谷胱甘肽还原酶表达及改变氧化还原酶活性通路来调节胞内氧化还原状态，通过上调脂肪酸、不饱和脂肪酸和甘油脂合成通路为细胞提供更多的脂肪酸、不饱和脂肪酸和油脂，缓解表面活性剂对细胞的不利影响，并通过上调橙色素合成基因（mrpigF）表达提高橙色素生成，为黄色素生成提供更多的前体；基于同源重组方法构建了mrpigF缺失菌株，与野生型菌株相比，ΔmrpigF菌株萃取发酵和常规发酵不生成橙色素和新黄色素，主要只生成黄色素monascin和ankaflavin，结合橙色素饲喂等实验进一步证实新黄色素来源于橙色素的酶催化反应。项目研究结果可以为萃取发酵相关机制研究及色素代谢调控奠定理论基础。

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