Multi-omics analysis reveals the temporal dynamics of enzymatic and microbial drivers in shaping ester synthesis during Nongxiangxing Daqu fermentation

Abstract

Daqu is the fermentation starter of Nongxiangxing Baijiu and plays a crucial role in flavor ester formation. However, the relationships among ester-forming capacity, ester-synthesizing enzymes, and functional microbial succession in Daqu remain poorly understood. Here, metagenomics, metaproteomics, and 16S rRNA/ITS amplicon sequencing were integrated to systematically characterize the dynamic interactions among ester-synthesizing enzymes, functional microorganisms, metabolic pathways, and flavor ester production throughout Daqu fermentation. Four key ester-synthesizing enzyme groups, including alcohol dehydrogenases (ADHs), alcohol acyltransferases (AATs), esterases (ESTs), and Baeyer-Villiger monooxygenases (BVMOs), exhibited distinct temporal expression patterns that were positively correlated with ester-forming capacity. Specifically, ESTs were predominantly involved in the formation of ethyl hexanoate, ethyl butyrate, and ethyl lactate during the middle and late fermentation stages, whereas AATs mainly contributed to ethyl acetate production from the early stage. ADHs supported ester biosynthesis by supplying alcohol precursors, while BVMOs showed limited involvement. Multi-omics analyses identified Lichtheimia and Rhizopus as major detected contributors to ester-synthesis-related enzyme profiles, whereas thermotolerant filamentous fungi, including Aspergillus and Rasamsonia, were enriched during mid-to-late fermentation and coincided with increased enzyme expression. KEGG analyses revealed a shift from substrate degradation and precursor generation to flavor-related metabolism, accompanied by a shift in the composition of functional microbial groups from Mucoromycetes-associated populations toward a consortium of thermotolerant filamentous fungi. Furthermore, solid-state fermentation confirmed the high ester-forming potential of Aspergillus niger. Collectively, this work provides a comprehensive characterization of the microbial and enzymatic dynamics associated with ester formation during Daqu fermentation, offering insights into the functional factors underlying ester synthesis and potential resources for improving ester-producing strains.

Keywords

Daqu / Ester-synthesizing enzymes / Ester synthesis / Metaproteomics / Solid-state fermentation / Nongxiangxing Baijiu

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Funding

the National Key Research and Development Program of China(2022YFD2101201)

National Natural Science Foundation of China(32072162)

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Jiangnan University