Table_3_Yeast Assimilable Nitrogen Concentrations Influence Yeast Gene Expression and Hydrogen Sulfide Production During Cider Fermentation.XLSX

The fermentation of apple juice into hard cider is a complex biochemical process that transforms sugars into alcohols by yeast, of which Saccharomyces cerevisiae is the most widely used species. Among many factors, hydrogen sulfide (H2S) production by yeast during cider fermentation is affected by yeast strain and yeast assimilable nitrogen (YAN) concentration in the apple juice. In this study, we investigated the regulatory mechanism of YAN concentration on S. cerevisiae H2S formation. Two S. cerevisiae strains, UCD522 (a H2S-producing strain) and UCD932 (a non-H2S-producing strain), were used to ferment apple juice that had Low, Intermediate, and High diammonium phosphate (DAP) supplementation. Cider samples were collected 24 and 72 h after yeast inoculation. Using RNA-Seq, differentially expressed genes (DEGs) identification and annotation, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, we found that gene expression was dependent on yeast strain, fermentation duration, H2S formation, and the interaction of these three factors. For UCD522, under the three DAP treatments, a total of 30 specific GO terms were identified. Of the 18 identified KEGG pathways, “Sulfur metabolism,” “Glycine, serine and threonine metabolism,” and “Biosynthesis of amino acids” were significantly enriched. Both GO and KEGG analyses revealed that the “Sulfate Reduction Sequence (SRS) pathway” was significantly enriched. We also found a complex relationship between H2S production and stress response genes. For UCD522, we confirm that there is a non-linear relationship between YAN and H2S production, with the Low and Intermediate treatments having greater H2S production than the High treatment. By integrating results obtained through the transcriptomic analysis with yeast physiological data, we present a mechanistic view into the H2S production by yeast as a result of different concentrations of YAN during cider fermentation.

苹果汁发酵制备硬苹果酒（hard cider）是一类复杂的生化过程，酵母可将果汁中的糖类转化为醇类物质，其中酿酒酵母（Saccharomyces cerevisiae）是应用最为广泛的酵母菌种。在诸多影响因素中，苹果酒发酵过程中酵母产硫化氢（hydrogen sulfide, H₂S）的能力受到酵母菌株以及苹果汁中酵母可同化氮（yeast assimilable nitrogen, YAN）浓度的共同调控。本研究旨在解析YAN浓度对酿酒酵母产H₂S过程的调控机制。本研究选用两株酿酒酵母：UCD522（产H₂S菌株）与UCD932（不产H₂S菌株），分别对添加低、中、高浓度磷酸二铵（diammonium phosphate, DAP）的苹果汁进行发酵。分别于酵母接种后24小时与72小时采集发酵中的苹果酒样品。本研究通过RNA测序（RNA-Seq）技术进行差异表达基因（differentially expressed genes, DEGs）的鉴定与注释，并开展基因本体论（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析，结果显示基因表达水平受酵母菌株、发酵时长、H₂S生成情况及其三者间交互作用的共同影响。针对产H₂S菌株UCD522，在三种DAP处理组中共鉴定得到30个特异性GO条目。在鉴定得到的18条KEGG通路中，“硫代谢”“甘氨酸、丝氨酸与苏氨酸代谢”以及“氨基酸生物合成”通路显著富集。GO与KEGG分析均显示“硫酸盐还原序列（Sulfate Reduction Sequence, SRS）通路”显著富集。此外，本研究还发现H₂S生成与应激响应基因间存在复杂的调控关联。针对UCD522菌株，本研究证实YAN浓度与H₂S生成量间呈非线性关系：低、中浓度DAP处理组的H₂S生成量均高于高浓度处理组。本研究将转录组分析结果与酵母生理数据相结合，阐明了苹果酒发酵过程中不同YAN浓度下酵母产H₂S的分子调控机制。