Abstract: This study aimed to elucidate the synergistic metabolic mechanism of a caproic acid-producing microbial community from pit mud of nongxiangxing baijiu. Metagenomics, metatranscriptomics, and metaproteomics were integrated to systematically investigate the succession of microbial community structure, the expression of functional genes, and their correlation with caproic acid synthesis during the fermentation process. The results showed that lactic acid was rapidly consumed in the early stage, acetic acid accumulated continuously, butyric acid increased rapidly in the early stage and then stabilized in the middle stage, and the caproic acid yield rose rapidly in the early and middle stages, reaching 14.53 g/L, and stabilized in the late stage. The fermentation process could be divided into three stages: adaptation (0-2 h), rapid synthesis (2-12 h), and stationary (12-24 h). The rapid synthesis of caproic acid relied on the highly expressed reverse β-oxidation system in the inoculum; the substrates were almost exhausted at the stationary stage, leading to the cessation of caproic acid production, and the microbial community underwent reprogramming of lipid metabolism, carbohydrate metabolism, and other pathways to adapt to substrate deficiency. A total of 12 core functional genera were detected by multi-omics, including Anaerococcus, Pseudoramibacter, Xylanivirga, Eubacterium, Peptoniphilus, Clostridium, Caproiciproducens. Species functional contribution analysis, metabolic pathway analysis and Spearman correlation analysis showed a precise functional division of labor among the microbial community: Pseudoramibacter, Eubacterium and Tetragenococcus were responsible for converting acetyl-CoA into acetoacetyl-CoA and other intermediates to provide precursors for carbon chain elongation; Peptoniphilus predominantly produced butyryl-CoA and butyric acid; Anaerococcus, Xylanivirga, Garciella, Clostridium and Finegoldia jointly undertook the core catalytic reaction extending butyryl-CoA to hexanoyl-CoA and the function of product efflux, serving as the executors of caproic acid synthesis; Caproiciproducens synthesized caproic acid independently through the fatty acid biosynthesis pathway. Together, they formed a metabolic network with a clear functional division of labor. This study revealed the temporal succession pattern and metabolic functional network of the caproic acid-producing microbial consortium from a multi-omics perspective, providing a solid theoretical basis and data support for an in-depth understanding of the microbial synergistic mechanism of caproic acid biosynthesis and the precise regulation of the fermentation process.

Key words: pit mud; caproic acid; mixed microbial system; microbial synergy; multi-omics