Microenvironmental Gradients Drive Spatial Stratification of Saccharifying Microbial Communities and Enzyme Activity in Strong-Flavor Daqu Fermentation

<i>Daqu</i>, a representative solid-state fermentation product, produces saccharifying enzymes to degrade sorghum starch into fermentable sugars for ethanol synthesis. Spatial heterogeneity in <i>Daqu</i> drives community assembly. However, its regulatory role in enzyme-driven saccharification remains unclear. By integrating metagenomics and PacBio full-length sequencing, this study investigated how microenvironmental gradients across distinct <i>Daqu</i> layers (QP (surface layer), HQ (middle layer), QX (center layer)) shape saccharifying microbiota and activity. Saccharifying activity exhibited a declining surface-to-center gradient (e.g., QP: 870.9 ± 21.2 U/mL > HQ: 631.2 ± 16.4 U/mL > QX: 296.5 ± 16.1 U/mL on day 30, <i>p</i> < 0.05), paralleled by divergence in microenvironments. Metagenomics identified α-amylase and α-glucosidase as key saccharifying enzymes, primarily encoded by fungi; their abundance was inhibited by heat and humidity, yet promoted by acidity. Enzymatic validation confirmed higher saccharifying activity in QP and HQ core microbes (e.g., <i>Lichtheimia ramosa</i>: 43.16 ± 1.97 U/mL) than in QX (e.g., <i>Paecilomyces variotii</i>: 14.27 ± 1.25 U/mL). Network analysis revealed Lactobacillaceae are closely linked with saccharifying communities. This study establishes microenvironmental gradients as critical regulators of spatial saccharification in <i>Daqu</i>, informing strategies to optimize microbial consortia for <i>baijiu</i> production.