Redox balance in wine yeast and the relation to hyperosmotic stress and wine quality

葡萄酒酵母中的氧化还原平衡及其与高渗应激和葡萄酒品质的关系

• 批准号: RGPIN-2020-05198
• 负责人: Inglis, Debra
• 金额: $ 2.04万
• 依托单位: Brock University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744765
• 关键词: Redox; balance; wine; yeast; relation

Icewine is a sweet dessert wine of critical importance to the Canadian wine industry and for international trade. Concentrated solutes (sugars and salts) in Icewine juice place wine yeast under hyperosmotic stress, leading to reduced cell growth, altered metabolism, 10-fold higher acetic acid, 2-fold higher glycerol, fermentation difficulties and sometimes organoleptic faults in wine. New wine styles for Canada are now made from dried grapes (appassimento wines), resulting in concentrated juices with similar issues. Fermentation problems are becoming increasingly relevant in common table wine production due to increased sugar in fruit due to global warming. Saccharomyces cerevisiae wine yeast display properties and DNA sequences distinct from well-studied S. cerevisiae laboratory yeast, and these differences impact yeast performance during fermentation and wine organoleptic properties. It is critical to understand these differences in selecting wine yeast best suited for high Brix fermentations. Intracellular accumulation of glycerol is essential for cell survival when yeast are subjected to hyperosmotic stress. Glycerol accumulation is accomplished through uptake of glycerol from the growth media via Stl1p, closing the glycerol export channel Fps1p, and increasing metabolic production from dihydroxyacetone phosphate. When cells produce glycerol internally, the NAD+ produced must be reduced to NADH by metabolite formation to maintain cellular redox balance. Acetic acid formation by yeast may be this mechanism but it is not clear which cofactor system its production is linked to, NAD+ or NADP+ via aldehyde dehydrogenases. While gene expression analysis is a useful tool for identifying potential gene targets associated with a given phenotype, further investigation is required to confirm protein function and phenotypic impact, which is lacking in the study of wine yeast under hyperosmotic stress. Constructing engineered wine yeast strains would be optimal for studying hyperosmotic stress during fermentation given the differing responses of laboratory and wine yeast strains and difficulties in separating isozymes from cellular compartments along with similar cofactor requirements. The central goal of my research program is to gain a comprehensive understanding of factors influencing wine yeast stress responses during fermentation of concentrated grape juices and how these responses relate to cellular redox balance, glycerol and organic acid homeostasis and wine quality. We will further advance this goal by confirming there is no glucose repression of membrane transporter STL1 in wine yeast under sugar-induced osmotic stress; determine if increasing [NAD+] while wine yeast are under hyperosmotic stress leads to increased acetic acid production by adding exogenous nicotinamide riboside; and quantifying roles of Ald 2-6p, alone and in combination, during wine fermentations by constructing wine yeast deletion mutants of ALD genes using CRISPR-Cas9.

冰酒是一种甜的甜点酒，对加拿大葡萄酒工业和国际贸易至关重要。冰酒汁中浓缩的溶质（糖和盐）使葡萄酒酵母处于高渗压力下，导致细胞生长减少，代谢改变，乙酸含量增加10倍，甘油含量增加2倍，发酵困难，有时还会导致葡萄酒的感官缺陷。加拿大的新葡萄酒风格现在由干葡萄制成（appassimento葡萄酒），导致浓缩果汁有类似的问题。由于全球变暖导致水果含糖量增加，发酵问题在普通餐酒生产中变得越来越重要。酿酒酵母的特性和DNA序列与实验室研究的酿酒酵母不同，这些差异影响了酵母在发酵过程中的表现和葡萄酒的感官特性。在选择最适合高糖度发酵的葡萄酒酵母时，了解这些差异是至关重要的。当酵母受到高渗胁迫时，细胞内甘油的积累是细胞存活所必需的。甘油的积累是通过Stl1p从生长介质中摄取甘油，关闭甘油输出通道Fps1p，增加二羟丙酮磷酸的代谢产量来完成的。当细胞内部产生甘油时，产生的NAD+必须通过代谢物的形成还原为NADH，以维持细胞氧化还原平衡。酵母生成醋酸可能是这种机制，但不清楚它的产生与哪个辅助因子系统有关，是通过醛脱氢酶产生的NAD+还是NADP+。虽然基因表达分析是识别与特定表型相关的潜在基因靶点的有用工具，但需要进一步研究以确认蛋白质功能和表型影响，这在高渗胁迫下的葡萄酒酵母研究中是缺乏的。考虑到实验室酵母菌和酒酵母菌对高渗胁迫的不同反应，以及从细胞区室分离同工酶的困难，以及相似的辅因子需求，构建工程酒酵母菌株是研究发酵过程中高渗胁迫的最佳选择。我的研究项目的中心目标是全面了解在浓缩葡萄汁发酵过程中影响葡萄酒酵母应激反应的因素，以及这些反应与细胞氧化还原平衡、甘油和有机酸稳态和葡萄酒质量的关系。我们将进一步推进这一目标，证实糖诱导的渗透胁迫下酒酵母中膜转运蛋白STL1没有葡萄糖抑制；通过添加外源性烟酰胺核苷，确定在葡萄酒酵母处于高渗胁迫下增加[NAD+]是否会导致醋酸产量增加；利用CRISPR-Cas9构建葡萄酒酵母Ald基因缺失突变体，定量分析Ald 2-6p单独或联合在葡萄酒发酵过程中的作用。