3.4 Engineering strategies to optimize the YESS system.
With any new engineering platform, several opportunities remain to improve the system through host cell and circuit engineering. The YESS system controls enzyme activities at the transcriptional and post-translational levels. Practically, achieving this control requires optimizing promoter strengths and testing ERSs with various retention strengths on the protease and substrates. Due to its lack of modularity, the original YESS plasmid faced a bottleneck when constructing plasmids to optimize activity and expression. Furthermore, enzyme transcription could not be turned off or controlled using a bidirectional GAL promoter. Titrating enzyme transcription is important to modulate enzyme: substrate stoichiometry in the ER, and the ability to turn off the enzyme is crucial in protease profiling experiments. Since cleaved substrates are selected for by a loss of fluorescence signal, any mutation in the substrate cassette’s C-terminal tag will appear as a false positive on FACS. To counter these challenges, Denard and coworkers tackled two aspects of the YESS system in YESS 2.0 (Denard et al., 2021). First, they used a two-step golden gate approach to enable rapid assembly of YESS plasmid parts, marking a first step towards a fully modular YESS platform (YESS 2.0). Second, they introduced a synthetic transcription factor in the EBY100 kex2Δ strain, enabling titratable β-estradiol induction of the protease, thus achieving decoupled transcription (Figure 3C). To showcase this advancement, they further engineered a TEV protease variant (eTEV) with an 8-fold faster catalytic efficiency than wild-type TEVp. Because YESS 2.0 could achieve low enzyme: substrate ratios in the ER, this evolved TEV showed a 3-fold increase in catalytic turnover.
Engineering the contact time between an enzyme and its substrate in the ER influences enzyme activity. Mei and coworkers studied how ERS sequences engaged ER receptors ERD1 and ERD2 (Mei et al., 2017). Their investigation discovered that the phenylalanine residue in the FEHDEL ERS sequence played a significant role in ERS: ERD2 interactions. By performing saturation mutagenesis at this residue, they discovered that the non-natural ERS sequence WEHDEL exhibited the strongest affinity for ERD2. Using this ERS on both the enzyme and substrate cassettes, they established activity for matrix metalloprotease 7 (MMP7) on an IgG-derived hinge sequence PAPELLGG, a previously intractable activity in the YESS system. This new ERS sequence now opens the possibility of engineering IgG-hinge cleaving metalloproteases. These improvements to the YESS system place this platform at the forefront of many biochemical assays of PTM-enzymes.