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.