SNARE基因在红曲色素转运中的作用机制研究

Monascus pigments(Mps), one of the main secondary metabolites produced by Monascus spp., are widely used as food additives in recent years which have many advantages, like stability, security and better coloring effect. However, the additional value of the Mps products is limited since the current Mps products are mixture, which are mainly used as colorant. Besides, the Mps productivity is very low and the single Monascus pigment was difficult to be obtained. The SNARE(Soluble-N-ethylmaleimide-sensitive fusion protein attachment protein receptor) genes (Mos2 and Mos7) involved in vesicle fusion and Mps transportation were discovered in our previous study. The gene deletion and overexpression mutants will be constructed to analyze their effect on the vesicles quantity and morphology, and the varieties and yield of intracellular and extracellular Mps. Furthermore, the transcriptional level of the genes involved in the Mps transportation will be analyzed to reveal the relationship between the vesicle fusion efficiency and Mps transportation after compared with the property of Ypt7 (regulated the fusion of vesicle and vacuole) deletion and overexpression mutants which were constructed before. In short, all these results will contribute to illustrate the relevance of Mps transportation mechanism and Mps biosynthesis pathway, improve the single Monascus pigment productivity and provide the theoretical guidance to the construction of genetical modified Monascus strain with high-producing Monascus pigments ablity.

红曲色素（Mps）是红曲菌（Monascus spp.）的主要次级代谢产物之一，具有稳定、安全、着色效果好等优势，是近年来应用广泛的食品添加剂。目前Mps产品均为混合物，主要用于食品着色，附加值不高，且生产效率较低，单一组分Mps难以制备，制约了Mps相关应用研究。本项目将围绕申请人前期发现与囊泡融合和Mps转运紧密相关的SNARE蛋白(可溶性N-乙基马来酰亚胺敏感性因子附着型的蛋白受体)编码基因Mos2和Mos7，通过基因敲除和过表达技术构建突变株，分析SNARE基因对囊泡数量、形态、胞内外Mps种类和含量影响的规律，通过与前期已构建的介导囊泡和液泡融合的Ypt7基因敲除和过表达突变株相比较，探讨SNARE基因对Mps转运关键基因转录进程的影响，阐明囊泡融合效率与Mps转运的相互关系。研究结果有望揭示Mps转运和合成途径的联系，提升单一组分Mps的发酵产率，指导高产Mps工程菌株的构建。

红曲色素（Monascus pigments, MPs）是我国传统药食同源真菌红曲菌（Monascus spp.）产生的一类次级代谢产物的总称，作为食品着色剂在我国已有两千年的应用历史。但现有的MPs产品均为混合物，产品用途单一，附加值不高，加之MPs的生产效率较低，极大地制约了MPs产品的应用研究。系统研究MPs的转运途径，揭示MPs转运过程和合成过程的联系，有望通过指导高产MPs工程菌株的构建来提升单一组分MPs的发酵产率。本项目通过分析申请人前期发现的与囊泡融合和MPs转运紧密相关的SNARE蛋白(可溶性N-乙基马来酰亚胺敏感性因子附着型的蛋白受体)编码基因Mos2和Mos7的功能，揭示其在MPs转运过程中的作用。首先通过基因敲除和过表达等分子生物学技术构建Mos2和Mos7基因的敲除、回补和过表达突变株共6株，并分析比较了不同突变株的菌落及菌丝形态、胞内外MPs种类和含量差异。并进一步通过与前期已完成的介导囊泡和液泡融合的Ypt7基因敲除和过表达突变株相比较，最后结合荧光定量PCR和转录组测序分析了Mos2和Mos7基因对红曲菌不同培养时期的与MPs合成和转运相关基因转录水平的影响，进一步阐明Mos2和Mos7基因在MPs转运中的功能。结果表明，Mos2基因参与红曲菌的氮源代谢，其缺失引起了MPs胞内积累；Mos7基因不影响红曲菌生长和产孢，其缺失在引起MPs的含量上升同时CIT的含量下降，其SNARE复合体的作用位点可能是MPs和CIT的转运分支节点。项目研究为系统揭示MPs的转运机制奠定了良好的数据基础，为指导高产MPs工程菌株的构建，提升单一组分MPs的发酵产率提供了新的思路。