1.2 Protease engineering on the yeast surface requires a unique approach.
Assaying bond-forming enzymes on the yeast surface is practical because these reactions yield a product with a labeled moiety. In contrast, engineering proteases in a similar manner to sortases is not a straightforward assay since proteolytic cleavage results in signal loss. To circumvent this obstacle and enable protease engineering and substrate profiling directly on the yeast surface, Hollfelder and coworkers developed a bait-and-capture YSD approach using the bacterial macromolecule alpha-2-macroglobulin (A2M) (Knyphausen et al., 2023). A2M is a naturally occurring broad-spectrum protease inhibitor. The mechanism of inhibition relies on a protease bait region and a buried receptor binding domain (Figure 1C). When a protease cleaves the bait region, an A2M conformational rearrangement results in the physical trapping of the protease and the exposure of a reactive thioester bond which may result in the formation of a covalent bond between A2M and protease lysine residues. Hollfelder showed that an active target protease on the yeast surface could be trapped by an A2M protein modified to present the protease’s target sequence, A2Mcap. This approach allowed them to evolve an SpIB variant that does not require removal of an N-terminal pro-sequence for activation, generating an enzyme that can more easily integrate synthetic biology and other applications. Lastly, a SCHEMA-based subdomain shuffling library of SplA-F followed by MACS and FACS selections led to a chimeric scaffold that supports specificity switching via subdomain exchange. Out of the 7 subdomains, subdomain 5 was found to confer most of the substrate specificity in Spl chimeras. Therefore, a diversification strategy focused on block 5 may be a good starting point for substrate specificity engineering in the Spl scaffold. Moreover, A2M capture is mechanism agnostic, meaning that it could be applied to many proteases and drive forward protease engineering for therapeutic applications.
Proteolysis-mediated protein activation.
Proteases with bespoke specificities are essential reagents in biotechnology and could usher in a new wave of enzyme therapeutics. Moreover, the principles of proteolysis-mediated nuclear translocation of transcription factors or protease-mediated activation of a toxic protein, common in protease-based synthetic circuits (H. K. Chung & M. Z. Lin, 2020), are used to build high-throughput screening and selections in yeast. To construct a protease-based transcriptional output, one can sequester a transcription factor away from the nucleus by confining it to the cytoplasm or attaching it to the inner plasma membrane via a linker harboring a protease-cleavage sequence. Once cleaved by the protease of interest, the transcription factor localizes to the nucleus to activate a reporter or an antibiotic resistance gene.
This concept was first illustrated with the Genetic Assay for Site-specific Proteolysis (GASP) system (Sellamuthu et al., 2011). Here, a lexA-b42 transcription is fused to the Ste2 transmembrane domain via a peptide linker containing a polyQ sequence found in the Huntingtin protein (Figure 2A). This setup was used to engineer the substrate specificity of Hepatitis A virus 3C Protease to cleave polyQ. Active site saturation mutagenesis isolated mutants around the S2 and S1’ subsites with improved activity towards polyQ. While these mutants largely showed relaxed rather than switched specificity, the Var26 variant prevented polyglutamine-induced neuronal cell death. Ting and coworkers built upon this concept to establish a significantly more advanced strategy to engineer proteases using blue light-induced protein-protein complementation between CRY and CIBN (Sanchez & Ting, 2020). Their design anchors a fusion protein composed of STE2Δ-CIBN-LOV-ProteaseCleavageSite-VP16 to the inner membrane (Figure 2C). The protease is expressed in the cytosol as a C-terminal fusion to the CRY protein. Under blue light, the CRY and CIBN proteins interact, bringing the protease closer to its substrate. Furthermore, access to the substrate is only possible because the LOV domain undergoes a conformational change, exposing the protease cleavage site. Protease cleavage releases the VP16 transcription factor, allowing it to migrate to the nucleus to activate the transcription of a fluorescent protein. Optogenetic protein circuits provide increased temporal resolution from seconds to hours, allowing one fine control over protein-protein interactions. This approach was applied to engineering TEV proteases with increased catalytic activity. After several rounds of evolution and selection stringency manipulations, Ting evolved high-affinity TEV proteases, including truncated TEV variants, uTEV1Δ, and uTEV2Δ, with turnover rates over 5-fold higher (compared to WT TEVΔ) and a full-length TEV protease, uTEV3 (2.5-fold faster than the WT enzyme). The increased catalytic efficiency of truncated variants improved SPARK (Kim et al., 2017) and FLARE (Wang et al., 2017), two transcription-based time-gated transcriptional readouts previously established by the Ting lab. Integration of new variants into these systems resulted in increased temporal control (uTEV1Δ responding to stimuli in <1 minute in SPARK, compared to the original design requiring >10 minutes) and overall enzymatic performance (uTEV1Δ observed to have increased signal-to-noise ratios 27-fold higher than original FLARE system).
A common limitation of protease-activatable transcriptional activation for protease engineering is the lack of a counterselection substrate. When evolving proteases for switched specificity, selecting against undesired cleavage sequences is imperative to avoid variants with relaxed specificity. Tucker and coworkers aimed to overcome this limitation by designing a proteolysis-dependent transcription factor inspired by hormone-inducible synthetic TFs (Cleveland et al., 2022). Their best design incorporated a counterselection substrate in the following fusion protein: estrogen receptor ligand binding domain-Gal4 binding domain-counterselection substrate-VP16 activation domain-selection substrate-estrogen receptor ligand binding domain, abbreviated as ER-LBD-GAL4BD-CS-VP16-SS-ER-LBD (Figure 2B). Without protease activity, this fusion protein resides in the cytosol as the ER-LBD remains bound to cytosolic Hsp90. Protease cleavage of the CS separates GAL4BD from VP16, resulting in an inactive transcription factor. Conversely, cleavage of the SS leads to the removal of one ER-LBD. This activity is enough to translocate the ER-LBD-GAL4BD-CS-VP16 to the nucleus, where it can activate a GAL-driven URA3 marker, leading to cell survival on Sc-URA plates. This approach was applied to evolve the cleavage specificity of Botulinum Neurotoxin Serotype B1 proteases (BoNT/B) away from its native sequence (VAMP3) towards an orthogonal substrate, specifically a VAMP3 sequence with a mutation at residue 59. They co-transformed a structure-guided triple mutant NNK library with 14 VAMP3 Q59 mutants. After URA3 selection, five mutants were obtained, including the promising RRG variant, which showed 10% cleavage of WT VAMP3, but 75% cleavage of VAMP3(Q59Y). However, the stringency of the counterselection cannot be tuned independently from the selection substrate, lowering the dynamic range this strategy can achieve.
Yeast ER sequestration accurately quantifies PTM-enzyme catalytic turnovers.
Engineering PTM-enzymes on the yeast surface or through transcriptional ON switches can suffer from design constraints that limit the extent to which PTM-enzyme catalytic properties can be modified. Transcription turn-on systems may require significant optimizations to establish a high dynamic range since a single protease turnover turns on a TF that catalyzes transcription exponentially. Similarly, a single turnover of a PTM-enzyme on the yeast cell surface is enough to generate a fluorescent signal. Therefore, in these platforms, increases in catalytic efficiency are driven mainly by improvements in substrate binding affinity. In cases where an increase in catalytic turnover (kcat ) is the goal, there is a need for a modified approach. In this vein, two technologies have been developed that leverage ER sequestration to engineer PTM-enzymes for multiple turnovers: the Yeast Endoplasmic Reticulum Sequestration and Screening (YESS) and the FRET-based protease evolution via cleavage of an intracellular substrate (PrECISE) systems. ER sequestration approaches, particularly ones based on the YESS approach, can give fine-tuned control in protein investigations.