黄酒酵母中β-苯乙醇合成代谢调控机理解析

β-phenylethanol with a rose-like fragrance is the most important aromatic alcohol in cooking wine and vinegar. The β-phenylethanol in cooking wine and vinegar is mainly from Chinese rice wine. The deficient regulation information about β-phenylethanol biosynthetic pathway hampers adjusting β-phenylethanol concentration in related products, which also limits the quality improvements of cooking wine and vinegar and perplexes the manufacturing enterprise. We have found that the β-phenylethanol in these products was produced by Chinese rice wine yeast which was named as Saccharomyces cerevisiae CRWY. However, there are little reports about the regulation mechanism of β-phenylethanol biosynthetic pathway in S. cerevisiae CRWY. Besides, the present reports and metabolic models about typical S. cerevisiae could not accurately descript the metabolic characterization of S. cerevisiae CRWY due to metabolic diversities at the subspecies level of S. cerevisiae. This study focused on the S. cerevisiae CRWY which is the functional microorganism of CRW, cooking wine and vinegar. By using the technologies of molecular biology, the molecular regulation mechanism of β-phenylethanol biosynthetic pathway in S. cerevisiae CRWY will be systematically revealed at genetic level, transcription level and enzymatic level. The expected results of this study will be helpful in solving the common and key problem about aroma enhancement of cooking wine and vinegar, and will provide the thesis for improving β-phenylethanol concentration in related products. The research achievements will be also useful in upgrading brewing technology and stably improving product quality. This will also push the “supply-side structural reform” in relative traditional brewing industry.

β-苯乙醇具有典型的玫瑰芬芳,是料酒、食醋中最重要的芳香醇。料酒及食醋中的β-苯乙醇主要来自于黄酒原料，目前关于黄酒中β-苯乙醇合成代谢调控信息缺乏，阻碍了相关产品中β-苯乙醇浓度的调节，限制了产品品质提升，困扰着生产企业。申请人研究发现黄酒酵母是相关产品中β-苯乙醇的主要来源，但目前鲜有关于黄酒酵母β-苯乙醇合成代谢调控机理的报道，由于酿酒酵母亚种间复杂的代谢多样性，现有代谢网络模型也不能够准确反应我国特色菌株——黄酒酵母的代谢特性。本课题以黄酒、料酒、食醋酿造的核心微生物黄酒酵母为研究对象，通过分子生物学手段从基因水平、转录水平和酶活水平三个层面整体揭示黄酒发酵过程中β-苯乙醇合成代谢调控机理。预期成果将有助于解决料酒及食醋增香的共性关键科学问题，为增加产品中β-苯乙醇含量提供理论依据，对于改进酿造技艺、提升产品品质和推动料酒及食醋行业的“供给侧”改革具有重要意义。

β-苯乙醇具有典型的玫瑰芬芳，是料酒、食醋中最重要的芳香醇。料酒及食醋中的β-苯乙醇主要来自于黄酒原料，目前关于黄酒中β-苯乙醇合成代谢调控信息缺乏，阻碍了相关产品中β-苯乙醇浓度的调节，限制了产品品质提升，困扰着生产企业。在自然科学基金项目支持下，本研究以我国特色酿造菌株--黄酒酵母为研究对象，通过分子生物学手段从基因水平、转录水平和酶活水平三个层面整体揭示黄酒发酵过程中β-苯乙醇合成代谢调控机理。发现相比于酿酒酵母模式菌株W303，黄酒酵母HJ生成β-苯乙醇的能力提高了50%，HJ的β-苯乙醇合成基因中24个编码区突变导致20个蛋白突变，22个有调控区域突变。调控区突变使得黄酒酵母Ehrlich途径关键基因的启动子强度提高了15%~100%，但莽草酸途径的大部分关键基因的启动子强度弱于模式菌，说明黄酒酵母具有更高活力的Ehrlich途径。转录水平分析也证实黄酒中β-苯乙醇主要来自于前酵期(120h内)的Ehrlich途径，发酵72h（生成了>60%的β-苯乙醇）内主要参与基因为GAP1、BAT2、PDC5、ADH1与ADH6，72-120h内主要参与基因为ARO3、ARO1、ARO2、ARO7、PHA2、ARO10、PDC5、ADH1与ADH2。首次发现预苯酸脱水酶（PHA2p）受到L-苯丙氨酸反馈抑制，并且突变体PHA2pHJ活力提高了22.6%，β-苯乙醇合成途径中其它酶突变使得酶活力提升了10%~25%。基于理论研究成果，从提高原料蛋白利用率、提高莽草酸途径通量、改变氮源种类、选育低醇高酯酵母等方面设计了β-苯乙醇调控策略，并进行了工业应用推广，β-苯乙醇最高浓度可达530 mg/L，是对照组的8.28倍，β-苯乙醇最低浓度可以降低30%，满足了料酒、食醋等调味品高β-苯乙醇浓度需求和黄酒等饮料酒的低β-苯乙醇浓度需求。本研究解决了料酒及食醋增香、黄酒降低杂醇的共性关键科学问题，对于改进酿造技艺、提升产品品质和推动传统酿造行业的“供给侧”改革具有重要意义。