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.