Abstract: [Objective] This study was conducted to characterize the change of functional gene expression of active microorganisms in different fermentation stages of koumiss. [Method] The koumiss samples were collected at the early （12 h）, middle （48 h） and late （96 h） stages of fermentation, and the metatranscriptomic libraries were prepared for high-throughput RNA sequencing. After assembly and functional annotation of the sequencing data, the differentially expressed genes （DEGs） were identified to determine the static enrichment of significant DEGs in GO and KEGG signaling pathways. [Result] The koumiss samples of the early, middle and late stages of fermentation generated 12.17 GB clean reads by metatranscriptomic high-throughput sequencing. Sequence assembly and data analysis demonstrated that the expression of genes involved in metabolic process played a dominant role in the fermentation process of koumiss. The identification and assessment of DEGs exhibited that the number of the down-regulated genes was more than that of the up-regulated genes in pairwise comparison of the koumiss samples collected at the three fermentation stages. GO functional enrichment analysis showed that the GO terms highly enriched by DEGs between the samples of early and middle fermentation stages associated with cellular process, metabolic process, organic ring compound binding, and heterocyclic compound binding. The GO terms highly enriched by DEGs between the samples of middle and late fermentation stages included cellular process and cellular macromolecule metabolic process. The GO terms highly enriched by DEGs between the samples of early and late fermentation stages mainly involved cellular process, organic substance metabolic process, nucleoside binding, ribonucleoside binding, and purine nucleotide binding. KEGG pathway enrichment analysis displayed that there was only one signaling pathway （ribosome） in which DEGs between the samples of early and middle fermentation stages significantly enriched. DEGs between the samples of middle and late fermentation stages significantly enriched in the signaling pathways of ether lipid metabolism, aminobenzoate degradation, mRNA surveillance pathway, and steroid biosynthesis. The signaling pathways of steroid biosynthesis, aminobenzoate degradation, mRNA surveillance pathway, ribosome and ethylbenzene degradation were significantly enriched by DEGs between the samples of early and late fermentation stages. [Conclusion] The differential expression of functional genes of active microorganisms was involved in the fermentation process and dynamically changed in different fermentation stages of koumiss. The results obtained in this study provide references for in-depth understanding the fermentation mechanism and optimizing the fermentation process of koumiss.

Key words: koumiss, fermentation, metatranscriptomics, functional gene