Abstract: To investigate the metabolic mechanism of esterase-producing Pichia kudriavzevii in mixed-culture fermentation, this study carried out tandem mass tag (TMT)-based quantitative proteomic analysis on P. kudriavzevii JM5-4 under pure culture and co-culture with Clostridium butyricum GD1-1. The metabolites of pure culture and co-culture were analyzed using gas chromatography-mass spectrometry (GC-MS). The results demonstrated that in mixed-culture fermentation, the yields of ethanol, acetic acid, and ethyl acetate were 2.396, 0.425, and 0.544 g/L, respectively, which increased by 1.5, 4.0, and 2.0 times when compared with monoculture fermentation respectively. Through TMT-based quantitative proteomics, a total of 3 164 quantifiable proteins were identified. Among these, 355 differentially expressed proteins (DEPs) were detected, out of which 159 were up-regulated and 196 were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses revealed significant alterations in the localization of proteins associated with tricarboxylic acid cycle, drug metabolism, small molecule metabolism, cytoplasmic large ribosomal subunits, cytoplasmic ribosomes, nicotinamide adenine dinucleotide binding, and oxidoreductase activity. The subcellular localization of the DEPs was analyzed using the online software CELLO, which assigned 257 proteins to eight distinct organelles, mainly distributed in the cytoplasm (129), mitochondria (91) and nucleus (19). KEGG annotation showed that these 355 DEPs were involved in metabolic subsystems such as oxidative phosphorylation, cofactor biosynthesis, the tricarboxylic acid cycle, and glycolysis/gluconeogenesis. During mixed-culture fermentation, all DEPs related to glycolysis/gluconeogenesis (e.g., pyruvate dehydrogenase and aldehyde dehydrogenase), tricarboxylic acid metabolism (including citrate synthase, aconitase, fumarase, and malate dehydrogenase), and glyoxylate and dicarboxylate metabolism (such as hydroxyl pyruvate reductase and aldehyde acid reductase) were up-regulated, indicating a significant enhancement in the growth and metabolism of JM5-4. Furthermore, DEPs associated with esterases (such as S-formyl glutathione hydrolase, ribonuclease, and trehalose phosphate phosphatase) were significantly up-regulated, suggesting that mixed-strain fermentation positively influenced the esterase-producing capacity of JM5-4. This study provides theoretical guidance for understanding the mechanism of mixed-strain fermentation in baijiu brewing and the metabolic characteristics of strains.

Key words: baijiu, Pichia kudriavzevii, tandem mass tag-based quantitative proteomics, metabolic products, esterase