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.