1.1 Directed evolution of bond-forming enzymes.
Since its initial development in 1997, yeast surface display (YSD) has
been a staple technology for studying, measuring, and evolving
biomolecular recognition (Gai & Wittrup, 2007; Raeeszadeh-Sarmazdeh &
Boder, 2022). YSD is attractive because many eukaryotic proteins can be
functionally expressed, secretory pathway engineering and protein
expression optimizations can achieve surface display of complex proteins
and assemblies (Lamote et al., 2023), and surface-displayed phenotypes
can be selected by fluorescence-activated cell sorting (FACS) or
magnetic-activated cell sorting (MACS). The most mature YSD method is
the AGA1p-AGA2p system, which takes advantage of the naturally occurring
disulfide bonded interactions between the a-agglutinin 1 and 2 subunits
on the yeast cell surface. Originally developed for engineering protein
binding affinity, YSD has been modified in several ways to measure,
control, and evolve enzyme activities (reviewed extensively elsewhere).
However, assaying PTM-enzymes on the yeast surface poses specific
challenges compared to other biocatalysts. First, proteases,
transglutaminases, and sortases, among others, are often synthesized as
inactive zymogens, requiring post-translational activation. Expressing
PTM-enzymes in their active forms can be toxic if they modify essential
host proteins. Second, pro-sequences can sometimes be necessary for
enzyme folding (Chang et al., 2018), and their activation may depend on
other enzymes or conditions not present in the yeast secretory pathway
(Zhao et al., 2014). Lastly, since PTM-enzymes react on proteins and
peptides and may require two or more substrates, it is necessary to
deliver these substrates in a way that safeguards the
genotype-to-phenotype linkage.
Creative designs circumvent and, at times, leverage these conditions to
engineer bond-forming enzymes on the yeast surface, particularly
sortases. The common strategy involves co-localizing a sortase and one
of its substrates on the yeast surface and exogenously supplying the
second substrate, which carries a handle for fluorescence detection or
magnetic selection (Figure 1A). For example, one can fuse a sortase and
its substrate to the 5’ and 3’ end of the aga2 gene, respectively
(Lim et al., 2017). When supplied with an azide-functionalized primary
amine nucleophile, sortase conjugates this molecule to its sorting
signal LPETGG. A copper-free Click reaction between the incorporated
azide and biotin-DBCO and subsequent labeling with streptavidin
conjugated to phycoerythrin allows them to visualize sortase activity on
the yeast surface by flow cytometry.
A more elaborate strategy to engineer bond-forming enzymes on the yeast
surface (Chen et al., 2011) involves fusing the enzyme to the C-terminus
of AGA2p, and connect an S6 peptide to the C-terminus of AGA1p (Chen et
al., 2011). The S6 peptide reactive handle is a substrate for Sfp
phosphopantetheinyl transferase from Bacillus subtilis , which
catalyzes the transfer of a CoA-activated peptide to the S6 peptide on
the yeast surface (Figure 1B). Liu and coworkers developed this system
to engineer Staphylococcus aureus sortase A (SrtA) variants with
increased activity on the native substrate LPETGG. In this case, a
biotinylated oligoglycine nucleophile is supplied to yeast cells
expressing sortase A variants and an S6 peptide-attached LPETGG
substrate, allowing sortase variants to perform the transpeptidation
reaction. This method obtained evolved sortases with up to 140-fold
increased catalytic efficiencies in LPETG-coupling reactions. These
variants showed significant improvements in target recognition and
kinetics of LPETGG-containing CD154 on the surface of HeLa cells. In a
subsequent study, S. aureus sortase A was evolved towards
non-canonical substrates LAXTG and LPXCG, resulting in two variants with
51,00-fold (eSrtA(2A-9), LAXTG) and 120-fold (eSrtA(4S-9), LPXCG)
switches in specificity (Dorr et al., 2014). Importantly, thekcat/KM of the two eSrtA variants
were comparable to that of the original eSrtA on LPXTG. This shows that
even under single turnover conditions, sortase directed evolution is
possible on the yeast surface.
More recently, Liu took the bold step of engineering S. aureussortase A to recognize an LMVGG sequence found in amyloid-β, which
differs from the canonical LPXTG at three positions (Podracky et al.,
2021). Starting from a variant with relaxed specificity (4S.6), they
tailored a directed evolution scheme that included lowering the
substrate concentration, altering selection conditions, and introducing
various decoy off-target substrates. The resulting variant SrtAβ,
obtained after 16 rounds of evolution, contained an additional 25
mutations compared to 4S.6 and exhibited over a 1,400-fold change in
substrate preference from LPESG to LMVGG. This modified preference
toward this target substrate provides the basis for using sortase
variants, particularly SrtAβ, as potential targeting agents to prevent
protein aggregation and related neurological diseases.
To engineer microbial transglutaminases (mTGs) and mitigate cytotoxicity
associated with their active forms, Kolmar and coworkers developed a
method to display an inactive mTG followed by activation via synthetic
pro-sequence cleavage (Deweid et al., 2018). mTGs can crosslink a side
chain of glutamine and primary amine (usually the ε-amino group of
lysine) via a transamidation reaction and are useful for site-specific
protein conjugations (Dickgiesser et al., 2019). To enhance the activity
of mTG, a synthetic pro-mTG was fused to the N-terminus of AGA2p. Once
on the surface, mTG could be activated with an enterokinase. When
supplementing a biotinylated glutamine-donor peptide, the activated mTG
could use its surface lysines as acyl acceptors, catalyzing their
labeling, thus enabling the selection of tagged cells by FACS (Figure
1D). This method could isolate mTG variants with up to 36% increased
catalytic efficiencies. One could argue that this method also selected
for mTG variants that remain stable with their surface lysines
increasingly modified.