弱化酿酒酵母乙醇合成能力的全局调控研究

Reducing the alcohol level in wine has become a major concern of the wine industry. Engineered Saccharomyces cerevisiae strain with reduced ethanol synthesis capacity, which would offer faster and less expensive biological alternatives to the current physical processes available for producing low- and reduced-alcohol wines. Currently, widely adopted methods, such as single (or multiple) gene knockout and overexpression, are not only difficult to markedly reduce the ethanol synthesis capacity of Saccharomyces cerevisiae, but also result acetic acid and acetaldehyde increased. In order to overcome the above deficiencies, in this project, Saccharomyces cerevisiae BY4742 was used as a research model, to study how weakening ethanol synthesis capacity of Saccharomyces cerevisiae by global regulation. Global transcription machinery engineering based on random mutation of the transcription factor gene SPT15, was employed to engineer Saccharomyces cerevisiae yeast strains for reducing ethanol synthesis capacity and also decreasing acetic acid and acetaldehyde production. Transcriptome and metabonomics technology in conjunction with intracellular NADH and NAD+ concentration detection, were used to elucidate the physiological mechanism of reduced ethanol synthesis capacity in Saccharomyces cerevisiae, in terms of NADH-NAD+ balance and carbon metabolism. The mechanism presented here, would give important theoretic foundation and provide new regulatory targets, for further weaken the ethanol synthesis capacity in Saccharomyces cerevisiae. Also, it may provide a theoretical basis for engineering microorganisms to optimize the synthesis of alcohol compounds.

开发低醇葡萄酒已成为现代葡萄酒工业的热点。构建乙醇合成能力弱化的酿酒酵母菌株是最为简单和经济的解决方法。目前所普遍采用的单纯通过单(多)基因的敲除或过量表达方法，不仅难以显著降低酿酒酵母乙醇合成能力，还会导致乙酸和乙醛等代谢副产物增加。为克服以上缺陷，本项目以酿酒酵母BY4742为研究模型，利用基于转录因子Spt15p的全局转录机器工程技术，构建乙醇合成能力弱化且乙酸和乙醛合成量下降的工程菌株。借助转录组学和代谢组学技术并辅以胞内NADH和NAD+浓度检测等，从NADH-NAD+平衡和碳代谢的角度阐释酿酒酵母乙醇合成能力弱化的生理机制。研究结果为系统改造酿酒酵母，进一步削弱乙醇合成能力奠定重要的理论基础和提供新的调控靶点，同时也对代谢工程改造微生物优化醇类化合物合成提供理论基础，具有重要的理论和实践意义。

开发低醇葡萄酒已成为现代葡萄酒工业的热点。构建乙醇合成能力弱化的酿酒酵母菌株是最为简单和经济的解决方法。 本项目以酿酒酵母YS59 为研究模型，利用基于转录因子Spt15p的全局转录机器工程技术，构建乙醇合成能力弱化的工程菌株，并利用组学技术手段分析其乙醇合成能力弱化的原因。研究结果表明，Spt15p上Ile 46 Met、Asp 56 Gly、Ser 118 Pro、Tyr 195 His、Leu 205 Ser等5个氨基酸的突变可使得酿酒酵母乙醇产率降低了37%。但对上述单个氨基酸进行突变反而会使乙醇产率有所提高，这说明乙醇产率的降低是多个突变氨基酸共同作用的结果。细胞内的还原力水平并不是乙醇合成能力弱化的决定因素。负责糖转运的HXK1/2基因显著下调表达，甘油途径的关键基因GPD1/2和GPP1/2均上调表达，谷氨酸与谷氨酰胺代谢途径产物显著增加，这些均导致甘油合成增加，从而减少乙醇合成。此外，参与NAD+合成的犬尿氨酸途径相关代谢物都有所增加，暗示碳源向TCA途径流动增强，从而也减少了用于乙醇合成的碳源。本项目研究结果为系统改造酿酒酵母，进一步削弱乙醇合成能力奠定重要的理论基础和提供新的调控靶点，同时也对代谢工程改造微生物优化醇类化合物合成提供理论基础，具有重要的理论和实践意义。