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.