Temporal dynamics and metabolic potential of microbial dark matter during dry storage of high-temperature Daqu revealed by metagenome-assembled genomes.

Abstract

High-temperature Daqu is the core starter culture for Jiang-flavor Baijiu production, whose microbial community directly shapes the enzymatic properties and flavor precursor profiles of fermentation systems. Dry storage drives microbial succession and functional maturation of Daqu, yet existing studies have predominantly focused on cultivable and well-characterized taxa, leaving the temporal dynamics and metabolic contributions of microbial dark matter (MDM) largely unexplored at the genome-resolved level. Using genome-resolved metagenomics, we reconstructed 52 medium- to high-quality metagenome-assembled genomes (MAGs) from high-temperature Daqu samples across a six-month dry storage period and identified five novel unclassified MDM lineages comprising four Bacillota-affiliated bacterial lineages and one archaeal lineage. Comparative genomic analysis revealed distinct functional differentiation between MDM lineages and dominant culturable taxa, as MDM lineages were enriched in genes associated with carbohydrate uptake and fermentative acid production and harbored a streamlined biosynthetic gene cluster (BGC) profile with uniformly high sequence novelty. Temporal dynamic analysis showed that MDM lineages from different storage microhabitats exhibited divergent succession patterns, and conventional physicochemical parameters showed limited explanatory power for MDM community variation, with only glucoamylase activity displaying a marginal association trend. This study provides genome-resolved insights into uncultured MDM lineages during high-temperature Daqu dry storage, expands the genomic repertoire of uncultured microbiota in traditional fermentation ecosystems, and offers novel perspectives for deciphering Daqu maturation mechanisms and quality regulation.