A microbial platform for selective biosynthesis of monoterpene esters

Reconstructing the precise biosynthesis of structurally complex natural esters, such as monoterpene esters, in engineered microbes remains a major challenge, owing to the limited repertoire of highly selective alcohol acyltransferases and the lack of compatible pathway modularity. Here, we establish a dual-substrate microbial platform to profile the activities of alcohol acyltransferase (AAT) to synthesize three distinct classes of monoterpene esters: monoterpenyl esters, monoterpenoate esters, and monoterpenyl monoterpenoate esters, enabling access to both natural and non-natural monoterpene ester biosynthetic pathways. Through structure-guided critical residue engineering and dual-substrate molar ratio tuning, we achieve selective biosynthesis of >C2 acyl-CoA-derived monoterpene esters, despite competing intracellular acetyl-CoA. Coculture engineering further redistributed metabolic fluxes between acyl-CoA and alcohol precursors, yielding 11.50 g/L linalyl acetate and 3.16 g/L geranyl butyrate in 1-L bioreactor. This study expands the biosynthetic space of monoterpene esters and provides a versatile strategy to control AAT selectivity, offering a plug-and-play, scalable framework for ester biomanufacturing.