Deciphering and Engineering the Substrate Specificity of Protein-Modifying Enzymes

Protein-modifying enzymes (PMEs) are ubiquitous in biology, playing significant roles in initiating, regulating, and terminating cellular processes. The diversity and breadth of their substrate preference have been harnessed for a variety of applications in biotechnology and biomedicine. To optimize PMEs towards these applications, it is often necessary to engineer their catalytic properties and to profile their substrate specificities. In searching for desired activities in a large pool of variants, a high-throughput screening or selection system should ideally exhibit a broad operational range and a high dynamic range.

Here we describe YESS 2.0, a modular and customizable yeast endoplasmic sequestration screening system suitable for engineering and profiling the specificity of PMEs. By incorporating features to modulate gene transcription, as well as substrate and enzyme spatial sequestration within a versatile and seamless assembly method, YESS 2.0 achieves broad operational and dynamic range. To showcase YESS 2.0, we evolve a TEV protease variant (eTEV) with an 8-fold higher catalytic efficiency to obtain the fastest TEV protease variant to date. Second, we use YESS 2.0 coupled with NextGen Sequencing to profile the substrate specificity of insulin-degrading enzyme (IDE) and discover IDE substrates with increased activity compared to the prototypical insulin peptide. Lastly, we show for the first time that YESS 2.0 supports calcium-independent sortase-mediated ligations (SML) and confirm that residues directly C-terminal of the pentapeptide motif heavily influence the rate of SML. YESS 2.0 offers unmatched versatility in profiling and engineering the specificity of PMEs and should enable even more ambitious future undertakings.