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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740445
• 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.

冰酒是一种甜甜酒，对加拿大葡萄酒工业和国际贸易至关重要。冰酒汁中的浓缩溶质（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，乙酸增加10倍，甘油增加2倍，发酵困难，有时葡萄酒中的感官缺陷。加拿大的新葡萄酒风格现在是由干葡萄（appassimento葡萄酒）制成的，导致浓缩果汁出现类似问题。由于全球变暖，水果中的糖分增加，发酵问题在普通餐酒生产中变得越来越重要。酿酒酵母葡萄酒酵母显示的性质和DNA序列不同于研究充分的S。酿酒酵母实验室酵母，这些差异影响酵母性能在发酵和葡萄酒感官特性。在选择最适合高白利度发酵的葡萄酒酵母时，了解这些差异至关重要。 当酵母受到高渗胁迫时，胞内甘油的积累对于细胞的存活是必不可少的。甘油积累是通过Stl 1 p从生长培养基中摄取甘油，关闭甘油输出通道Fps 1 p，并增加磷酸二羟丙酮的代谢产物来实现的。当细胞内部产生甘油时，产生的NAD+必须通过代谢产物形成还原为NADH以维持细胞氧化还原平衡。酵母形成乙酸可能是这种机制，但不清楚其产生与哪种辅因子系统相关，NAD+或NADP+通过乙醛脱氢酶。虽然基因表达分析是一个有用的工具，用于确定潜在的基因靶点与给定的表型，需要进一步的调查，以确认蛋白质的功能和表型的影响，这是缺乏在高渗胁迫下的葡萄酒酵母的研究。鉴于实验室和葡萄酒酵母菌株的不同反应以及从具有相似辅因子要求的细胞室沿着中分离同工酶的困难，构建工程葡萄酒酵母菌株将是研究发酵过程中高渗胁迫的最佳选择。 我的研究计划的中心目标是获得一个全面的了解因素影响葡萄酒酵母应激反应在发酵过程中的浓缩葡萄汁，以及这些反应如何与细胞氧化还原平衡，甘油和有机酸的稳态和葡萄酒的质量。我们将进一步推进这一目标，通过确认在糖诱导的渗透胁迫下葡萄酒酵母中膜转运蛋白STL 1没有葡萄糖阻遏;确定在葡萄酒酵母处于高渗胁迫下时增加[NAD+]是否会通过添加外源性烟酰胺核苷导致乙酸产量增加;以及通过使用CRISPR-Cas9构建ALD基因的葡萄酒酵母缺失突变体来定量Ald 2-6p单独和组合在葡萄酒发酵期间的作用。