Genome editing to generate nonfoam-forming sake yeast strains

Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1∆/awa1∆ deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1∆/awa1∆ in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding. K7GE01, a nonfoam-forming sake yeast strain, was developed to have awa1Δ/awa1Δ deletion alleles and few unexpected mutations by genome editing tools.清酒酵母在育种过程中常发生突变，并产生意料之外的表型。本研究借助基因组编辑（genome editing）工具，开发得到一株理想的无泡型清酒酵母菌株K7GE01。该菌株携带有负责调控无泡表型的纯合awa1Δ/awa1Δ缺失等位基因，且脱靶突变（off-target mutations）极少。高维形态表型分析（high-dimensional morphological phenotyping）结果显示，该基因组编辑菌株与其亲本菌株未检出可观测的形态学差异；而经典无泡清酒酵母菌株K701则表现出显著的形态学变化。小规模发酵试验同样证实，K7GE01与K701所酿造清酒的成分存在差异。K7GE01所酿造的清酒，其乙酸乙酯（ethyl acetate）、苹果酸（malic acid）、乳酸（lactic acid）与乙酸（acetic acid）的浓度存在显著差异；而K701所酿造清酒的成分差异更为显著。本研究结果证实了清酒酵母分离株中awa1Δ/awa1Δ的真实表型，并证明了基因组编辑工具在清酒酵母育种中的应用价值。无泡型清酒酵母菌株K7GE01通过基因组编辑工具开发得到，其携带有awa1Δ/awa1Δ缺失等位基因且意外突变极少。