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

Our 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 used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the 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. Coaux-seq is a new method for high-throughput protein characterization, utilizing complementation of auxotrophs and next generation sequencing.The use of simple barcode sequencing (BarSeq) allowed for cost-effective and simple screening of 11 different genome fragment libraries across 20 different auxotrophic strains.The first experimental evidence of function is provided for 53 proteins.Unexpected functions were identified in three enzymes, including a sulfate uptake transporter from the TauE family, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase.Workflow optimizations and discussions on potential pitfalls are provided. Coaux-seq is a new method for high-throughput protein characterization, utilizing complementation of auxotrophs and next generation sequencing.