Figure legends
Figure 1. Leveraging Yeast Surface Display for PTM-enzyme
engineering. (A) Using AGA1-AGA2 surface binding proteins to tether
sortase variants and substrate targets to the yeast surface using N and
C terminal binding and screened by either FACS or MACS. (B) Selection of
tethered sortase enzymes first by the acetylation of the substrate
peptide (S6) sequence, a reaction that selectively activates the
tethered sortase. Cell populations are incubated with a nucleophile and
then stained with a specific fluorescent antibody for that nucleophile,
followed by selection by either FACS or MACS. (C) Protease variants
tethered to the N-terminus of AGA2 screened for cleavage efficiency of a
substrate sequence blocking protease trapping via A2M. Active proteases
are trapped, labeled with fluorescent markers, and screened by FACS. (D)
Screening of tethered mTG variants to determine the efficiency of
binding glutamine-donor peptides to free lysine, screened by FACS.
Figure 2. Transcription-based screens of engineered PTM-enzymes
via cytosolic sequestration. (A) Screening of active enzyme variants
against non-canonical substrates using a membrane-tethered transcription
factor (TF), tethered by desired substrate sequence, for growth-based
selection. B) Dual selection screening of enzyme efficiency and
cleavage-site recognition of substrate orthologs via TF tethered to
estrogen-receptor ligand binding domains (LBD) by substrate sequences.
(C) Engineered PTM-enzyme circuit in yeast that selects for active
engineered variants through blue light-mediated substrate exposure and
enzyme-substrate proximity facilitated by the recognition of the CRY and
CIBN domains
Figure 3. Yeast ER sequestration accurately quantifies
PTM-enzyme catalytic turnovers. (A) YESS enzyme (blue) and substrate
(purple) cassettes present on a plasmid that can be transformed into
yeast for enzyme-substrate activity assays, including signal peptides
(SP), ER retention signals (ERS), and unique epitope tags for
fluorescence staining. (B) YESS can perform library screening of (1)
protease variants, (2) substrate variants, or (3) both. Enzyme-substrate
interactions can be assayed for enzymatic activity, substrate
selectivity, and selectivity modulation using FACS, MACS, NGS, and ML.
(C) Two-promoter design of YESS 2.0 with ꞵ-estradiol induction driving
protease expression and substrate expression remaining under galactose
induction. (D) PrECISE: Selection of active variants against a desired
substrate motif within a large library achieved using a
protease-mediated protein FRET-based assay.