基于逆生物合成分析的红曲色素基因簇最小化研究

红曲色素（Monascus pigments, MPs）是红曲菌（霉）分泌的主要次生代谢产物之一，有上千年应用历史，也是近年我国增长最快的食品着色剂。MPs是包括红、橙、黄三类色素的混合物，由MPs基因簇表达的多个酶级联催化所得。尽管MPs的基因簇已报道，并采用基因敲除方法明确了其中少数基因的功能，但MPs合成途径仍不清楚。目前这种“基因—酶—产物”的正向思路研究合成途径，结果往往不理想，所以一种基于“产物—酶—基因”的逆生物合成分析方法成为研究热点。本项目拟以前期获得的MPs主要组分之一，红斑胺（rubropunctamine，RM）为切入点，根据其结构，通过逆生物合成分析推导合成RM所必需的酶，在基因簇中搜索对应基因；在克隆、表达、分析上述基因功能的基础上，构建由这些基因组成的最小色素基因簇，在米曲霉中异源表达，以阐明RM合成途径。研究将为解析MPs合成途径提供另一种思路。

Monascus pigments (MPs), one class of the most important secondary metabolites produced by Monascus spp., has been used for more than thousands of years. The demand for MPs as food colorant is rapidly increased in China. MPs, the mixture of red, orange and yellow constituents, are catalyzed by enzymes produced by Monascus pigment biosynthetic gene cluster. Although one document reported about the MPs biosynthetic gene cluster, only a few gene functions have been identified. Therefore, the biosynthetic pathways of MPs are still unclear. To date, the study on biosynthetic pathway of MPs has mainly focused on the common route “genes—enzymes—metabolites”, but its efficiency is low. Recently, another method which is named as retrobiosynthetic analysis, based on the inverse route “metabolites—enzyme—gene”, has caught the attention. This study firstly set rubropunctamine (RM), one of the MPs main constituent, as starting-point of retrobiosynthetic analysis, and selected the corresponding genes for biosynthetic pathway of RM. Secondly, we will clone those genes and then construct the recombinant Aspergillus oryzae for heterologous expression of enzymes. The catalytic function of these enzymes will be further studied. At last, we will minimize biosynthetic gene cluster of RM in A. oryzae, and analyze the structure of secondary metabolites, on the purpose of exploring the biosynthetic pathway of RM. This research may provide a new method for exploration of MPs biosynthetic pathway.