Substrate-mediated spatial cooperation in Bacillus-Saccharomyces consortia enhance starch hydrolysis and flavor formation during solid-state fermentation.

Solid-state fermentation is extensively applied in the production of fermented foods, bio-based chemicals, and bioenergy products. However, rational regulation of fermentation performance remains difficult because microbial succession and metabolic labor division are strongly influenced by spatially heterogeneous substrates. The mechanisms through which spatially organized metabolic interactions coordinate substrate conversion and product biosynthesis remain insufficiently understood. Here, using Daqu fermentation as a representative model, temporal transcriptomic analyses revealed a distinct metabolic succession pattern, characterized by Bacillus-dominated starch hydrolysis during the liquefaction stage and Saccharomyces-driven ethanol production during the subsequent fermentation stage. Fluorescently labeled Bacillus-Saccharomyces synthetic consortia further demonstrated that starch availability critically governs colony expansion and spatial cooperation. To achieve programmable control of starch degradation, an isopropyl β-D-1-thiogalactopyranoside-inducible α-amylase expression system was constructed, enabling precise regulation of starch hydrolysis kinetics, metabolic productivity, and the spatial organization of the consortium. In parallel, individual-based modeling simulations closely reproduced experimental observations and demonstrated that substrate-mediated metabolic interactions are key determinants of spatial cooperation in communities with division of metabolic labor. Application of the engineered consortium in Daqu fermentation significantly improved starch conversion efficiency and flavor compound formation, while simultaneously reshaping the indigenous microbial community structure. Collectively, these findings reveal that spatial cooperation between Bacillus and Saccharomyces on starch substrates is a key mechanism linking efficient starch utilization to enhanced flavor production, providing a foundation for the rational design of high-performance microbial consortia in spatially structured fermentation systems.