Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation

Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms. The first aim of this study was to assess the most competitive strains that grow under wine fermentation conditions at low temperature. To this end, we performed a growth competition assay with 27 commercial wine strains inoculated at equal population size in synthetic grape must. In spite of the economical and industrial importance of these strains, their phenotypic variation in the main enological traits, particularly those related to optimum growth temperature, and their ability to adapt to low temperature fermentation have been poorly investigated. The second goal was to obtain an improved strain to grow and ferment at low temperature by evolutionary engineering. For this purpose, we maintained growth competition in synthetic grape must during 200 generations to select for the mutations that produce phenotypes with improved growth in this medium. One of these evolved cultures was previously treated with ethyl methanesulfonate (EMS) to increase the mutation rate. Finally, we aimed to decipher the molecular basis underlying this improvement by analyzing the genomic and transcriptional differences between the parental strain and the strain evolved at low temperature.

低温酿造的葡萄酒通常被认为可提升感官品质。然而，低温发酵存在若干弊端：例如生长速率低下、延滞期较长，以及发酵迟缓或停滞。筛选并培育适配低温环境的新型酿酒酵母（Saccharomyces cerevisiae）菌株，对于未来的生物技术应用具有重要价值。
本研究旨在通过进化工程技术，筛选并培育能够良好适配低温发酵环境的葡萄酒酵母菌株，并解析所获表型背后的分子机制。为此，我们选取27株商业酵母菌株的混合菌群，设置批量连续稀释实验以模拟12℃下的葡萄酒发酵条件。进化工程借助酵母自然突变率与诱变流程完成。其中一株菌株（P5）在两种实验条件下均优于其他菌株，并在200次传代后成为优势菌群。
与原始亲本菌株相比，进化后的菌株生长性能与低温发酵能力均得到显著提升。该性能提升仅在肌醇限制培养条件下才可获得。对进化菌株与亲本菌株的转录组比较分析显示，四个隶属于DAN/TIR家族的甘露糖蛋白编码基因（DAN1、TIR1、TIR4与TIR3）的上调幅度最为显著。
对进化菌株的基因组测序发现，GAA1基因存在一处单核苷酸多态性（SNP）；在原始菌株的单倍体衍生物中构建定点突变体GAA1Thr108后，其发酵性能得到改善。GAA1基因编码糖基磷脂酰肌醇（GPI）转酰胺酶复合物的一个亚基，负责将GPI锚定修饰添加至新合成的蛋白质（包括甘露糖蛋白）中，而GPI锚定是肌醇合成所需的关键过程。
因此，本研究证实了肌醇与甘露糖蛋白在酵母低温适应中的重要性，并通过关联两种代谢途径阐明了GAA1基因的核心调控作用。本研究的首要目标是评估在低温葡萄酒发酵条件下生长竞争力最强的菌株。为此，我们将27株商业葡萄酒酵母以相同种群规模接种至合成葡萄汁培养基中，开展生长竞争实验。
尽管这些菌株具有重要的经济与工业价值，但针对其主要酿酒学性状的表型变异（尤其是与最适生长温度相关的性状），以及它们对低温发酵的适应能力，相关研究仍较为匮乏。本研究的第二个目标是通过进化工程技术，获得能够在低温环境下生长与发酵的改良菌株。为此，我们在合成葡萄汁培养基中维持生长竞争达200次传代，以筛选出可提升该培养基中生长性能的突变株。其中一株进化菌群曾经甲基磺酸乙酯（EMS）处理以提高突变率。最后，我们旨在通过分析亲本菌株与低温进化菌株的基因组与转录组差异，解析该性能提升的分子基础。