Mixed fermentation with Saccharomyces cerevisiae and Pichia kudriavzevii has been shown to enhance wine aroma, yet the underlying mechanisms remain unclear. Monoculture of S. cerevisiae, monoculture of P. kudriavzevii, and mixed culture of S. cerevisiae and P. kudriavzevii were conducted, and the study analyzed and compared the biomass, flavor profile, and transcriptome responses of the three groups. Both yeast species exhibited growth inhibition in mixed culture, especially P. kudriavzevii. Significant differences were observed in three organic acids and the foremost 20 volatile compounds. Mixed fermentation enhanced esters (e.g., ethyl butyrate, isoamyl acetate) and volatile acids (e.g., hexanoic acid), but decreased isobutanol, phenylethyl alcohol, and quinic acid. Transcriptomic analysis revealed 294 and 332 differentially expressed genes (DEGs) in S. cerevisiae and P. kudriavzevii, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation results indicated that DEGs in mixed fermentation were concentrated in carbohydrate metabolism and amino acid metabolism. Our integrated analysis suggested that genes such as TDH2, TDH3, and ENO2 were pivotal for ester biosynthesis. Moreover, ADH1, ADH2, HPA3, ALD6, and ARO8 were associated with quinic acid synthesis. Furthermore, ILV2, ILV5, ALD6, and others were central to the production of isobutanol and phenylethyl alcohol.

Feruloyl esterase (FAE)-producing micro-organisms to obtain ferulic acid (FA) from natural substrates have good industrial prospects, and the synergistic effect of multiple bacteria can better improve the yield of FA. In this study, on the premise of the synergistic effect of FAE, hemicellulose, and cellulase, the key strain Klebsiella oxytoca Z28 with FAE was combined with CMCase and Xylanase-producing strains to produce FA. The combination of strains with higher FA production are Klebsiella oxytoca Z28, Klebsiella pneumoniae JZE, Bacillus velezensis G1, and their FA production can reach 109.67 μg/g, which is 15% higher than that of single bacteria. To explore the effects of temperature, Ph, inoculum amount, distillers grains concentration and fermentation time on the FAE activity of the combination of strains in the fermentation process, and determined that temperature, Ph, and fermentation time were the main influencing factors and optimized through orthogonal design. The optimized fermentation conditions are 34 °C, Ph 8.0, and fermentation days for 6 days, the FAE activity can reach 270.78 U/L, and the FA yield of the combined strain is 324.50 μg/g, which is 200% higher than that of single-strain fermentation.