上面发酵酵母高级醇代谢网络分析与低产高级醇小麦啤酒工业菌株的选育

In previous study, we found that the genetic diversity of Saccharomyces cerevisiae and the mechanism of gene regulation of higher alcohols metabolism is not clear, resulting in gene transformation of industrial strains of Saccharomyces cerevisiae genome according to the standard, often failed to achieve the desired results, and sometimes even contrary results. During the fermentation of wheat beer the top-fermenting yeast Saccharomyces cerevisiae performs a high yield of higher alcohols, moreover, there are some troubles in the control of the production of higher alcohols in the top-fermenting yeast S. cerevisiae. Thus, understanding metabolic mechanism of higher alcohols in S. cerevisiae and breeding of excellent strains are therefore of great importance to both academic and industrial research. In this study, metabonomics combined with transcriptomics will be used to identify the differentially expressed genes in the metabolism of higher alcohols under different fermentation conditions. Construction of differential expressed gene mutations, analysis of the changes of mutant metabolic properties, combined with higher alcohols metabolism in KEGG in the analysis of top-fermenting yeast higher alcohols metabolic regulation mechanism, determine the main functional genes that affect the production of higher alcohols. In order to construct industrial S. cerevisiae strain without the presence of heterologous DNA, markerless deletion method of genes and gene seamless insertion technique will be used to control and optimize the metabolic network of higher alcohols in top-fermenting yeast. This step results in the less higher alcohols of the excellent industrial S. cerevisiae strains, as well as will provide coordinated change of a ratio of esters and higher alcohols and improve the quality of wheat beer. The trace metabolic regulation and breeding technology system of the higher alcohols produced by top-fermenting yeast industrial strains will be also established. This research can provides theoretical basis for the regulation of the trace amounts of metabolites produced by top-fermenting yeast industrial strains, and also will be of great importance in enhancing the production technology level and the international competitiveness of our country's alcoholic beverage industry.

课题组前期研究表明，由于酿酒酵母工业菌株的遗传多样性及高级醇代谢调控机理不明确等问题，导致按照酿酒酵母基因图谱对工业菌株进行基因改造时，往往达不到预期效果。本课题针对上面发酵酵母酿造小麦啤酒过程中高级醇类物质含量过高的问题，拟利用代谢组学与转录组学相结合的方式，研究上面发酵酵母不同发酵工艺条件下高级醇类物质生成量及代谢相关基因转录水平的变化，探明引起高级醇类物质代谢变化的差异基因；构建高级醇代谢表达差异基因突变株，分析突变株代谢性能的变化，结合已有的高级醇类物质代谢途径，解析上面发酵酵母高级醇代谢调控机理，确定影响上面发酵酵母高级醇生成的主要功能基因；采用基因无痕敲除技术及基因启动子无缝插入技术等，对影响高级醇代谢的主要功能基因进行改造，构建适量低产高级醇的上面发酵酵母工业菌株。研究结果将完善上面发酵酵母高级醇代谢调控机理，对提高我国酿酒工业的生产技术水平和国际竞争力具有重要意义。

小麦啤酒是以小麦芽或未发芽的小麦作为主要原料生产的一类啤酒，国际上，一般要求小麦芽用量占原料总量50%以上才称为小麦啤酒。目前，国内外生产啤酒所使用的原料主要是大麦麦芽，而我国国产大麦的产量远远满足不了国内啤酒工业发展的需要，近七成要从国外进口。同时，我国又是小麦产量最多的国家，如何使用小麦麦芽生产啤酒，充分利用我国丰富的小麦资源，开发高品质、个性化的啤酒产品，带动农业现代化的发展，增加农产品的附加值，是当前啤酒工业需要解决的重要课题。我国啤酒企业自上世纪 80 年代就已经开始试制小麦啤酒，但多年来小麦啤酒在消费者中的认知度并不高，发展缓慢，小麦啤酒高级醇含量高，饮后会产生口渴、头痛等症状，不利于饮用者的身体健康，严重影响了小麦啤酒的发展。针对上面发酵酵母酿造小麦啤酒过程中高级醇类物质含量过高的问题，本项目利用代谢组学与转录组学相结合的方式，研究了上面发酵酵母不同发酵工艺条件下高级醇类物质生成量及代谢相关基因转录水平的变化，探明了引起高级醇类物质代谢变化的差异基因；成功构建高级醇代谢表达差异基因突变株，通过分析突变株代谢性能的变化，结合已有的高级醇类物质代谢途径，解析了上面发酵酵母高级醇代谢调控机理，最终确定了影响上面发酵酵母高级醇生成的主要功能基因。成功采用基因无痕敲除技术及基因启动子无缝插入技术等，对影响高级醇代谢的主要功能基因进行改造，构建了适量低产高级醇的上面发酵酵母工业菌株，研究结果完善了上面发酵酵母高级醇代谢调控机理。另外，本项目中对不同α-氨基氮浓度发酵条件下的酿酒酵母进行了转录组学和代谢组学分析，解析了酿酒酵母高级醇代谢调控机理，确定了α-氨基氮对酿酒酵母高级醇代谢的调控机制，本项目的研究成果对提高我国酿酒工业的生产技术水平和国际竞争力具有重要意义。

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