Aromatic esters are a class of versatile compounds with broad applications in the food, cosmetic, and pharmaceutical industries. Their diverse chemical structures determine their specific functionalities. Among them, aromatic ethyl esters are formed by esterification of aromatic acids and ethanol, which is traditionally synthesized via chemical methods under acidic and high-temperature conditions. In contrast, biosynthetic approaches offer a sustainable and mild alternative. In this study, we established the first artificial biosynthetic pathways for aromatic ethyl esters production in E. coli by introducing two-step heterologous enzymatic reactions. Firstly, heterologous enzymes were screened from enzyme databases based on their catalytic mechanisms and substrate promiscuities. Then, to improve heterologous enzyme activity, we engineered their substrate catalytic pockets by rational mutagenesis. Furthermore, E. coli’s endogenous metabolism was reprogrammed to direct carbon flux to aromatic precursors synthetic pathways. Finally, de novo production of multiple aromatic ethyl esters was achieved in shake flask by using glycerol as sole carbon source. This work showcased the capabilities of microbial cell factories in producing value-added aromatic ethyl esters through tailored artificial biosynthetic pathways.
