3.1 YESS for protease engineering.
The Yeast Endoplasmic Reticulum Sequestration and screening (YESS) system allows one to accurately control and quantify catalytic turnovers and select variants solely based on these activities (Figure 3A). The YESS system leverages yeast surface display in a clever way, where the activity of the PTM-enzyme can be measured on the yeast cell surface, while the enzyme remains in the ER. One can visualize the YESS system as a flow reactor with an enzyme anchored in the ER, and the substrate cassette, destined for cell surface display, is modified as it travels through the ER and secretory pathway. Once on the surface, substrate modifications can be visualized using fluorescently labeled antibodies. Cells harboring the desired modifications can be isolated by FACS or MACS. Originally reported ten years ago, the YESS system consists of two components. The first is to target and partially retain two or more transcriptional cassettes, one for the enzyme, and the other for the enzyme’s substrate(s), by appending an ER signal peptide to the 5’ and an ER retention sequence (ERS) to the 3’ end of each coding sequence, respectively. The second is to design a substrate cassette, typically fused to the C-terminus of AGA2p. The substrate cassette is the activity reporter and can be a short sequence flanked by epitope tags or a full protein. For protease engineering, YESS allows one to incorporate both counterselection and selection substrates, a vital attribute to avoid engineering protease generalists. Lastly, polypeptide retention in the ER can be manipulated by changing ERSs with weak or strong binding affinities to ER receptors. This way, the stoichiometry and contact time between an enzyme and its substrate can be readily titrated. While the YESS system presents many moving parts, it enables unprecedented control of ER-localized manipulation of enzyme activities at the transcriptional and post-translational levels.
In its initial development, YESS was used to evolve TEV proteases with orthogonal P1 specificities (Yi et al., 2013). Yi and coworkers employed a library-against-library screening approach, where an S1 pocket saturation mutagenesis of TEV protease and a substate cassette containing the native substrate as counterselection ENLYFQS and ENLYFXS as a selection substrate library (saturation mutagenesis at P1 position) were interrogated simultaneously. After several rounds of sorting and further error-prone engineering, screening, and analysis homed in on two TEVp protease variants, PE10 and PH21. TEV-PE10 and TEV-PH21 showed 5,000-fold and 1,100 switches in substrate specificity toward a P1 Glu and P1 His, respectively. Furthermore, the authors show that removing the ERSs from the substrate and protease cassettes allowed them to evolve a faster TEV protease on its canonical substrate, TEV-Fast, with a 4.6-fold increase in catalytic efficiency.