Genetic selection for small molecule production in competitive microfluidic droplets

Abstract

Biosensors can be used to screen or select for small molecule production in engineered microbes. However, mutations to the biosensor that interfere with accurate signal transduction are common, producing an excess of false positives. Strategies have been developed to avoid this limitation by physically separating the production pathway and biosensor, but these approaches have only been applied to screens, not selections. We have developed a novel biosensor-mediated selection strategy using competition between co-cultured bacteria. When applied to biosynthesis of cis,cis-muconate, we show that this strategy yields a selective advantage to producer strains that outweighs the costs of production. By encapsulating the competitive co-cultures into microfluidic droplets, we successfully enriched for muconate-producing strains from a large population of control non-producers. Facile selections for small molecule production will increase testing throughput for engineered microbes and allow for the rapid optimization of novel metabolic pathways.