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.