A Novel Screening System to Characterize and Engineer Quorum Quenching Lactonases

N‐acyl l‐homoserine lactones are signaling molecules used by numerous bacteria in quorum sensing. Some bacteria encode lactonases, which can inactivate these signals. Lactonases were reported to inhibit quorum sensing‐dependent phenotypes, including virulence and biofilm. As bacterial signaling is dependent on the type of molecule used, lactonases with high substrate specificity are desirable for selectively targeting species in communities. Lactonases characterized from nature show limited diversity in substrate preference, making their engineering appealing but complicated by the lack of convenient assays for evaluating lactonase activity. We present a medium‐throughput lactonase screening system compatible with lysates that couples the ring opening of N‐acyl l‐homocysteine thiolactones with 5,5‐dithio‐bis‐(2‐nitrobenzoic acid) to generate a chromogenic signal. We show that this system is applicable to lactonases from diverse protein families and demonstrate its utility by screening mutant libraries of GcL lactonase from Parageobacillus caldoxylosilyticus. Kinetic characterization corroborated the screening results with thiolactonase and homoserine lactonase activity levels. This system identified GcL variants with altered specificity: up to 1900‐fold lower activity for long‐chain N‐acyl l‐homoserine lactone substrates and ~38‐fold increase in preference for short‐chain substrates. Overall, this new system substantially improves the evaluation of lactonase activity and will facilitate the identification and engineering of quorum quenching enzymes.