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.

Icewine是对加拿大葡萄酒行业和国际贸易至关重要的甜品葡萄酒。冰川汁中的浓缩固体（糖和沙拉）将酵母放在过度渗透胁迫下，导致细胞生长减少，代谢改变，乙酸高10倍，甘油高2倍，发酵难度以及有时在葡萄酒中的器官断层。现在，加拿大的新葡萄酒风格是由干葡萄（Appassimento葡萄酒）制成的，导致浓缩果汁具有类似的问题。由于全球变暖，由于水果中的糖增加，发酵问题在普通餐桌葡萄酒中变得越来越相关。酿酒酵母酵母酵母显示特性和DNA序列不同于酿酒酵母的实验室酵母，这些差异会影响发酵过程中的酵母菌性能和葡萄酒有机充血的特性。了解这些差异在选择最适合高brix发酵的葡萄酒酵母方面的差异至关重要。当酵母承受过度渗透应激时，甘油的细胞内积累对于细胞存活至关重要。甘油的积累是通过通过STL1P从生长培养基中吸收甘油来实现的，关闭甘油导出通道FPS1P，并增加了二羟基苯乙酮的代谢产生。当细胞内部产生甘油时，必须通过代谢产物形成将产生的NAD+减少到NADH，以维持细胞氧化还原平衡。通过酵母菌形成醋酸盐可能是这种机制，但尚不清楚其生产与NAD+或NADP+通过醛脱氢酶相关的哪种辅助系统。虽然基因表达分析是识别与给定表型相关的潜在基因靶标的有用工具，但需要进一步研究以确认蛋白质功能和表型影响，这在高渗透应激下对葡萄酒酵母的研究缺乏。鉴于实验室和葡萄酒酵母菌菌株的不同响应以及将同工酶与细胞隔室分离，以及相似的辅助因素，构建工程葡萄酒酵母菌菌株对于在发酵过程中研究过度渗透应力是最佳选择。我的研究计划的核心目标是对影响浓缩葡萄汁发酵过程中影响葡萄酒酵母应力反应的因素进行全面了解，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。我们将通过确认在糖引起的渗透胁迫下，在葡萄酒酵母中膜转运蛋白STL1的葡萄糖表达进一步促进了这一目标。确定葡萄酒酵母在高渗性应力下是否增加[NAD+]是否会通过添加外源性烟酰胺核糖剂来增加乙酸的产生。并使用CRISPR-CAS9构建ALD基因的葡萄酒酵母缺失突变体，从而在葡萄酒发酵过程中单独和组合量化ALD 2-6p的作用。