Cross-kingdom metabolic cooperation drives vanillic acid biosynthesis: A spatiotemporal dissection of microbial functional networks in solid-state fermentation

Microbial self-organization into spatiotemporally structured consortia is key to metabolic specialization in natural environments, yet the principles governing this process in food fermentation are poorly understood. Here, we elucidate how cross-kingdom microbial cooperation drives the biosynthesis of vanillic acid (VA), a critical flavor and bioactive phenolic compound, during the solid-state fermentation of strong-flavor baijiu (SFB). Integrated metagenomic and network analyses across stratified pit layers and fermentation stages revealed a defined three-phase succession model. Early phase (D0-D12) was dominated by filamentous fungi (Aspergillus, Paecilomyces) in upper layers, initiating starch hydrolysis and phenylpropane precursor synthesis (e.g., contributing 22.6% to phenylalanine ammonia-lyase). A transitional bacterial-fungal consortium (Pichia, Klebsiella) then mediated intermediate conversion (D12-D45), with enzymatic hotspots shifting downward. The maturation phase (D45-D85) was defined by the dominance of acidophilic Acetilactobacillus (>80% relative abundance) in the lower layer, which executed the final synthesis steps (contributing 31.5% to caffeic acid O-methyltransferase) and concurrently suppressed vanillic acid degradation via downregulation of vanillate O-demethylase. Network analysis confirmed a spatial metabolic division of labor: fungi specialized in upper-layer lignin deconstruction, while bacteria dominated the completion of phenylpropanoid pathways in the lower layer. Critically, peak VA accumulation (0.375 mg/L at D45) coincided with synchronized enzyme expression across layers, demonstrating active metabolic coordination rather than passive environmental filtering. Our findings establish that functional succession and spatial compartmentalization are fundamental ecological principles enabling efficient biosynthesis in solid-state fermentation, demonstrating that flavor outcomes can be programmed through targeted microbial consortium design.