CRISPR-Cas9 Mediated UGA1 Knockout in Saccharomyces cerevisiae for Enhanced GABA Production in Wort Beverage

γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter, is known for its physiological functions in alleviating anxiety and improving sleep. Currently, high-yielding GABA food products are mainly obtained through screening wild-type high-producing strains (e.g.,Saccharomyces cerevisiaeisolated from Sichuan pickles yielding 0.67 g/L) or employing co-culture systems (e.g.,Enterococcus faeciumandLactiplantibacillus plantarumreaching 6.35 g/L). While effective, these methods often rely on natural screening strains or multi-microbial interactions. This study employed CRISPR-Cas9 technology to knockout theUGA1gene inSaccharomyces cerevisiae, a key gene responsible for GABA degradation. Starting from the low higher alcoholSaccharomyces cerevisiaeSY-LH, we successfully constructed the recombinant strain SY-LHU. Remarkably, this study discovered a significant upregulation ofGAD1gene expression followingUGA1knockout, which further enhanced GABA synthesis capacity. Under optimal fermentation conditions (inoculum size 4 × 107cells/mL, wort concentration 10 °P, sugar addition 60 g/L, 30 °C for 10 days, and mixing the malt broth every 48 h), the validation fermentation was performed and the GABA content in the wort beverage reached 280.36 mg/L, representing a 385.4% increase compared to the pre-optimization level. Furthermore, sensory evaluation by a trained panel yielded a mean score of 88, with no significant off-flavors detected, demonstrating the product’s high consumer acceptance. This pioneering work provides a novel and feasible technical pathway for developing functional alcoholic beverages with sleep-aiding properties.