利用转录组方法解析不同阶段红曲霉合成红曲色素、Monacolin K、桔霉素的代谢网络

Monascus produce a variety of secondary metabolites, such as Red Pigment as food coloring, the ideal drug for cholesterol-lowering Monacolin K, citrinin toxicity. The research showed that: the red pigment, Monacolin K, and citrinin are polyketides, the initial stage are similar in the biosynthetic pathway and accompanied produce, but the metabolic regulation mechanism of red pigment , monacolin K and citrinin are not clear. Therefore the study of the three substances metabolic network could help to achieve high efficiency, low toxicity, safety engineering strain theory and application basis. In this project, take the Monascus pilosus with untreated as research object, through physical chemical mutagenesis screening high-yielding red pigment, high yield Monacolin K, and low-yielding citrinin mutant strains. Three mutants and research object were analyzed by using RNA sequencing at different stages in order to found the differentially expressed genes. The function of these differentially expressed genes and five significant genes (mokA, mokE, mokC, mokF , mokH) of encoding functional domains on polyketide synthase were verified by using fluorescence quantitative PCR. The results can provide us a basis for construction the metabolic network for red pigment, monacolin K and citrinin in Monascus pilosus and cultivate the new engineering strain lay the foundation.

红曲霉产生多种次生代谢产物，如可作为食用色素的红曲色素、降低胆固醇理想药物的Monacolin K，具有毒性的桔霉素。研究表明：红曲色素、Monacolin K和桔霉素均为聚酮类化合物，在合成途径中起始阶段是相似的,且相伴产生，但其代谢调控机制尚不明确，对于三种物质的代谢网络研究可为实现高效、低毒、安全的工程菌株提供理论和应用依据。本研究以红曲霉Monascus pilosus为对照，通过诱变筛选高产红曲色素、高产Monacolin K、低产桔霉素的突变菌株，利用RNA-Seq技术分析四种菌株在高产红曲色素和Monacolin K不同阶段的基因表达谱，找到差异表达的关键酶基因，并对差异显著基因和聚酮体合成酶上的编码不同功能域的五个关键基因进行转基因功能验证，为解析红曲霉在不同阶段合成红曲色素、Monacolin K、桔霉素的代谢网络及构建新的工程菌株打下基础。

红曲霉产生多种次级代谢产物，如可作为食用色素的红曲色素、具有降胆固醇功效的Monacolin K，具有毒性的桔霉素等。研究表明：红曲色素、Monacolin K和桔霉素均为聚酮类化合物，在合成途径中起始阶段是相似的，且相伴产生，但其代谢调控机制尚不明确。本项目很好地完成了相关研究任务。具体研究成果为：（1）采用紫外-氯化锂复合诱变筛选获得高产色素菌株Y1，采用常压室温等离子体（ARTP）技术诱变红曲霉菌株M-1，筛选获得高产MonacolinK突变株23。（2）利用illumina的Hiseq2000高通量测序平台对构建Paired-End片段库进行样本测序，并进行基因组注释及表达量分析。（3）结合聚酮类化合物合成过程中的高产红曲色素和高产MonacolinK，构建 PKS 中的各个功能域的网络代谢途径，确认参与红曲色素、MonacolinK、桔霉素代谢合成相关的关键酶基因及调控基因。（4）在以转录组的高通量测序结果，以及MonacolinK表达量的定量分析结果为基础，构建mokC、mokD、mokE、mokI基因的过表达工程菌株菌株。本项目从转录组学的角度解析了红曲霉在不同阶段合成红曲色素、Monacolin K、桔霉素的代谢网络及构建新的工程菌株，以推动中国功能红曲产品的发展。本研究成果已有7 篇论文被录用，其中SCI 2 篇，核心期刊4 篇；培养硕士生 2名，按期完成了相关研究任务，达到了预期目标。