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Abstract

Strain breeding is essential for biomanufacturing, yet technological limitations and the scarcity of high-performance strains hinder progress. Beer brewing, as a typical food biomanufacturing process, particularly depends on strain quality. Although gene editing has advanced, engineered strains face food application restrictions, highlighting the urgent need for efficient, high-throughput screening methods. In this study, we investigated the underlying mechanisms limiting fermentation efficiency and identified glucose repression as the key barrier via phenotypic analysis and gene editing. We further confirmed that maltotriose utilization is a critical performance indicator. Using adaptive laboratory evolution (ALE), 2-deoxyglucose (2-DG) solid-phase screening, DWS-Dp3 screening (Dp3-Sole Substrate Deep-Well Plates Screening), and fermentation validation, we successfully obtained the industrial yeast strain ZPJ303–7, showing a 5% increase in fermentation degree and stable performance. Our findings elucidate the key mechanism restricting fermentation providing a practical high-throughput screening approach for strain improvement, offering valuable guidance for trait enhancement in related fields.