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, lead to high intoxicating degree (ID). To produce the low-ID whiskey, the Saccharomyces cerevisiae XF0-h, XF0-H and XF0-LH were successfully constructed by CRISPR-Cas9 gene editing technology to knock out HOM2 (encoding aspartate β-semialdehyde dehydrogenase) in the original strain XF0 and the LEU1 knockout strain XF0-L. The content of higher alcohols in whiskey fermented by the 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 the XF0, XF0, and XF0-L. The fermentation conditions of the XF0-LH were optimized through single-factor experiments and Box-Behnken design. The optimal conditions were the wort concentration of 9.8 °P, the hydrolyzed broken rice syrup addition of 78 g/L, and the inoculum size of 2.7 × 106 cells/mL. The low-ID whiskey was brewed with higher alcohols content at 556 mg/L by 50L fermenter at the optimal conditions.