(627a) A Modular, Broadly-Applicable Platform for High-Throughput Engineering and Profiling of Protein-Modifying Enzymes

Here we describe Modular YESS (MYESS), a highly versatile version of the yeast endoplasmic sequestration screening (YESS) system suitable for engineering and profiling the specificity of protein-modifying enzymes. By incorporating features to modulate gene transcription, as well as substrate and enzyme spatial sequestration within a versatile and seamless assembly method, MYESS achieves higher sensitivity and dynamic range. To showcase MYESS, we evolve a TEV protease variant (eTEV) with an 8-fold higher catalytic efficiency to obtain the fastest TEV protease variant to date. Due to the unique features of our system, this phenotype is strictly attributable solely to an increase in turnover number (k_cat). Second, we used MYESS coupled with NextGen Sequencing to profile the substrate specificity of insulin-degrading enzyme and discover IDE substrates with increased activity compared to the prototypical insulin peptide. Lastly, we show for the first time that MYESS supports calcium-independent sortase-mediated ligations and confirm that residues directly C-terminal of the pentapeptide motif heavily influence the rate of SML. The state-of-the-art MYESS platform offers unmatched versatility in profiling and engineering the specificity of protein-modifying enzymes and should enable even more ambitious future undertakings.