酿酒酵母酯醇代谢机理的研究与工业菌种的定向改造

Ester and higher alcohol play a key role in the sensorial quality of fermented alcoholic beverages like wine, beer and sake. During fermentation the yeast Saccharomyces cerevisiae produces very little esters and performs a high yield of higher alcohols. Moreover, there are some troubles in the control of the productions of flavour-active compounds, such as ethyl lactate, ethyl butyrate, ethyl caproate and n-propyl alcohol, in the yeast S. cerevisiae. Thus, understanding metabolic mechanism of higher alcohols and esters in S. cerevisiae and breeding of excellent strains are therefore of great importance to both academic and industrial research. In this study, the functions of EHT1, EEB1, IAH1, FAA1, BAT1, BAT2, THR4, ILV1, ILV2, LEU1, ADH2 and HOM2 and their interaction mapping will be identified. The differences in gene expression levels under different fermentation conditions and the influences of the differences on the metabolism of flavors will be monitored. And then the metabolic mechanism of higher alcohols and esters in S. cerevisiae will be better understanded. In order to construct industrial S. cerevisiae strain without the presence of heterologous DNA, an efficient system for promoter replacement and a seamless precise genome editing system will be constructed using a CRISPR-Cas system and a seamless gene deletion method through a two-step integration protocol constructed in our previous study. The metabolism of higher alcohols and esters in S. cerevisiae will be controlled and optimized. This step results in the more aroma production of the excellent industrial S. cerevisiae strains producing more esters and less higher alcohols, as well as will provide coordinated change of a ratio of esters and higher alcohols and improve quality of the wine product. The trace metabolic regulation and breeding technology system of the flavors produced by industrial S. cerevisiae strains will be also established. This research can provides theoretical basis for the regulation of the trace amounts of metabolites produced by industrial S. cerevisiae 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.

本课题针对酿酒酵母风味物质代谢酯低醇高的现象和乳酸乙酯、丁酸乙酯、己酸乙酯、正丙醇等风味物质代谢调控存在的问题，构建酿酒酵母酯醇代谢相关基因（产物）功能连锁网络，结合不同发酵工艺对酿酒酵母酯醇代谢相关基因表达差异的分析，完善酿酒酵母酯醇代谢的分子遗传机理；为保证构建的工业酿酒酵母菌株不带有存在安全隐患的外源基因，拟通过CRISPR-Cas系统结合本实验室前期开发的两步基因整合法的基因无痕敲除技术，建立高效、灵活的酵母基因启动子替换以及基因无痕精确编辑系统，并对工业酿酒酵母酯醇代谢网络进行精细调控和优化；选育出适量高产酯、低产高级醇的酿酒酵母工业菌株，使其在酿造过程中生成较多的风味物质且实现酯醇比例协调，促进酿酒行业的优质高产。研究结果将完善工业酿酒酵母风味物质代谢调控机理与育种技术体系，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。

酯和高级醇是构成如白酒、啤酒和清酒等饮料酒酒体和风味的重要成分，而酿酒酵母风味物质代谢存在酯低醇高的现象。本研究通过对醇乙酰基转移酶基因过表达与BAT2基因敲除协同作用，IAH1和BAT2基因敲除与ATF1基因过表达协同作用，ILV1、LEU1、LEU2基因参与的α-酮酸合成作用与分解作用，α-酮酸的脱羧作用对酿酒酵母酯和高级醇代谢的影响进行探讨，比较分析突变株和亲本株生长、发酵性能，酯和高级醇生成量，相关基因mRNA水平，全面深入的研究酿酒酵母酯和高级醇代谢的调控机制，完善了酿酒酵母酯醇代谢的分子遗传机理。研究了发酵过程中三种常见乳酸菌对高产酯酿酒酵母MY-15酯醇代谢的影响，探索了酿酒酵母与乳酸菌协同发酵的代谢机制。通过易错PCR构建了PGK1启动子突变文库，利用文库中的PGK1启动子构建敲除BAT2同时过表达ATF1基因的酿酒酵母重组菌株，对工业酿酒酵母酯醇代谢网络进行精细调控和优化。构建了一种快速、高效的无痕基因敲除系统（通过I-SCEI内切酶介导的DSB修复机制）；对无痕基因敲除系统进行了优化；并应用到低产高级醇高产酯酿酒酵母优良菌株的选育。研究结果完善了工业酿酒酵母风味物质代谢调控机理与育种技术体系，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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