Enhanced Mannan Production of <i>Saccharomyces cerevisiae</i> by CRISPR/Cas9 and Mannoproteins Characteristics on Wine Astringency Modulation

Mannoproteins are critical in modulating wine astringency, yet the specific impacts of their monosaccharide ratio and side-chain structure remain insufficiently explored. This study employed CRISPR/Cas9 to engineer yeast strains producing mannoproteins with either a high mannose-to-glucose ratio (high-yield-mannan strain BSFA12) or a nonbranched <i>N</i>-glycan structure (BY4741-ΔMNN2). The resulting mannoproteins (MP_BSFA, MP_BY2) were compared against controls (MP_BY extracted from <i>Saccharomyces cerevisiae</i> BY4741, a commercial product MP₆₀) using physicochemical analyses (fluorescence quenching, dynamic light scattering, and isothermal titration calorimetry) and sensory evaluation. Both engineered variants demonstrated superior astringency reduction in model and red wines. Our results establish that an increased mannose-to-glucose ratio and reduced <i>N</i>-glycosylation significantly enhance the astringency-mitigating effect, providing a foundation for the industrial-scale production and application of mannoprotein additives.