(629d) Discovery of New and Tunable N-Terminal Protein Degradation Tags in E. coli using FACS & NGS

The N-degron pathway canonically links a protein’s half-life to the identity of its N-terminal amino acid, serving as a protein quality control mechanism present throughout all domains of life. Notably, traditional methods for N-degron discovery and characterization have been limited by their low throughput. Here, we present a high-throughput in vivo degron screening platform that interrogates the substrate specificity of the Escherichia coli N-degron pathway by pairing a ratiometric fluorescent protein degradation assay with a fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS) workflow. Our workflow can evaluate the stabilizing/destabilizing effect of five simultaneously randomized N-terminal amino acids (P1-P5) with position-by-position resolution based on the enrichment present in sorted bins of varying fluorescence. We first optimized our ratiometric assay on a microplate reader to distinguish between known stabilizing and destabilizing degrons. We then identified the sequence specificity of native E. coli degradation machinery by simultaneously randomizing positions 2-5 for degrons starting with each of the canonical six destabilizing N-termini (L, F, Y, W, R, and K). We then obtained a greater understanding of the substrate specificities of the individual N-recognin proteins in the E. coli N-degron pathway by comparing enrichment of the same library expressed in knockout strains. We paired our in vivo screen with fluorescence anisotropy and computational docking to validate and explain our results. Overall, these results provide us with the highest resolution profiling of the E. coli N-degron pathway to date. Our screening workflow paired with the sequence stability exceptions that we observe provide a pathway for the design of synthetic degradation tags with increased predictability and tunability.