High-throughput protein characterization by complementation using DNA barcoded fragment libraries

Abstract

AbstractOur ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we usecomplementation ofauxotrophs and DNA barcodesequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains ofEscherichia colito identify genes that complement missing biochemical activity. Although assay effectiveness ranged with respect to source genome, with 41% of expected enzymes recovered, even distantE. colirelatives likeBacillus subtilisandBacteroides thetaiotaomicronshowed success. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein on an amino acid basis. Among unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq’s utility is demonstrated, with future applications in ecology, health, and engineering.