Investigating the main contributors to esterification activity and identifying the aqueous-phase ester synthases in Daqu

Fatty acid ethyl esters contribute desirable flavors for fermented foods , particularly Baijiu. The ester synthase is critical for the flavor ester synthesis, and Daqu , which serves as fermentation starter , is a primary source of the enzyme . However, the microbial source and ester synthase characteristics in Daqu remain poorly understood, limiting targeted strategies to enhance esterification . In this study, the esterifying microorganisms and their aqueous-phase ester synthase in Daqu were investigated based on full-length sequencing and molecular simulation methods. The filamentous fungi including Thermoascus aurantiacus , Aspergillus niger , A. chevalieri , A. flavus , Rhizopus microspores , and Byssochlamys spectabilis , were demonstrated as the main contributors to esterification activity. Using molecular dynamics simulation and molecular docking , the catalytic characteristic of known aqueous-phase ester synthase in A. niger was revealed. Based on the catalytic characteristic, a novel class of aqueous-phase ester synthase with a unique catalytic pentad (Tyr-His-Ser-His-Asp) was identified using molecular simulation method, which is widely present in filamentous fungi of Daqu , such as A. niger , A. chevalieri , A. flavus , R. microsporus , and B. spectabilis . Moreover, the unique catalytic structure could be used to identify potential aqueous ester synthase. Overall, this study enhances the understanding of esterifying microorganism and ester synthase in Daqu , and it is helpful for identification of esterifying microorganism and ester synthase in complex fermentation ecology. • The main stage of esterification activity formation in Daqu was clarified. • Filamentous fungi are main contributors to esterification activity for Daqu. • Molecular simulation reveals characteristics of aqueous-phase ester synthetase. • A novel class of ester synthase with a specific catalytic pentad was identified.