Engineering a SyntheticEscherichia coliCoculture for Compartmentalizedde novoBiosynthesis of Isobutyl Butyrate from Mixed Sugars

ABSTRACT Short-chain esters are versatile chemicals with use as flavors, fragrances, solvents, and fuels. The de novo ester biosynthesis consists of diverging and converging pathway submodules, which is challenging to engineer to achieve optimal metabolic fluxes and selective product synthesis. Compartmentalizing the pathway submodules into specialist cells that facilitate pathway modularization and labor division can present a promising solution. Here, we engineered a synthetic Escherichia coli coculture with the compartmentalized sugar utilization and ester biosynthesis pathways to produce isobutyl butyrate from a mixture of glucose and xylose. To compartmentalize the sugar-utilizing pathway submodules, we engineered a xylose-utilizing E. coli specialist that selectively consumes xylose over glucose and bypasses the carbon catabolite repression (CCR) while leveraging the native CCR machinery to activate a glucose-utilizing E. coli specialist. Upon compartmentalizing the isobutyl butyrate pathway submodules into these sugar-utilizing specialist cells, a robust synthetic coculture could be engineered to selectively produce isobutyl butyrate at a level of 392 mg/L, about 31-fold higher than the monoculture.