HOM2 Deletion by CRISPR-Cas9 in Saccharomyces cerevisiae for Decreasing Higher Alcohols in Whiskey

In typical whiskey, the content of higher alcohols is about 1500–2000 mg/L, leading to a high intoxicating degree (ID). To produce low-ID whiskey,Saccharomyces cerevisiaeXF0-h, XF0-H and XF0-LH were successfully constructed by CRISPR-Cas9 gene editing technology to knockoutHOM2(encoding aspartate β-semialdehyde dehydrogenase) in the original strain XF0 and theLEU1knockout strain XF0-L. The contents of higher alcohols in whiskey fermented by XF0-h, XF0-H, and XF0-LH were 704 ± 8 mg/L, 685 ± 6 mg/L, and 685 ± 19 mg/L, respectively, showing reductions of 23.93%, 25.98%, and 15.81% compared to XF0, XF0, and XF0-L. The fermentation conditions of XF0-LH were optimized through single-factor experiments and the Box–Behnken design. The optimal conditions were a wort concentration of 9.8 °P, hydrolyzed broken rice syrup addition of 78 g/L, and an inoculum size of 2.7 × 106cells/mL. The low-ID whiskey was brewed with a higher alcohol content of 556 mg/L by 50 L fermenter at the optimal conditions.