Introduction
Protein post-translational modifications (PTMs) largely regulate the
functional proteome. Thus, the enzymes that catalyze these
transformations (PTM-enzymes) are central to understanding cellular
processes in health and disease. Aside from their physiological roles,
PTM-enzymes are foundational to biotechnology and synthetic biology
because they catalyze chemo-, regio-, and sequence selective PTMs of
user-defined proteins and peptides. Synthetic PTMs can impart new
functions to biomolecules, introduce probes and labels, and establish
customized genetic and protein circuits in living cells. With their
diverse sequence specificities, PTM-enzymes enable technologies in
proteomics, biorthogonal chemistry, and biochemical and cellular
imaging. For instance, proteases with broad specificity have transformed
mass spectrometry-based proteomics (Giansanti et al., 2016; Schräder et
al., 2017; Tran et al., 2016). Contrariwise, proteases with narrow
specificity are heavily used in recombinant protein purification (Waugh,
2011), drive protein-based synthetic circuits in cells (H. Kay Chung &
Michael Z. Lin, 2020; Fink & Jerala, 2022; Gao et al., 2018), and are
being engineered as potential enzyme therapeutics (Shankar et al.,
2021). Transpeptidases such as sortases (Pishesha et al., 2018) and
asparaginyl endopeptidases allow one to generate multifunctional protein
conjugates that can serve as therapeutics and imaging agents (Morgan et
al., 2022). Finally, enzymes that modify specific amino acid side
chains, including ligases, transferases, oxidoreductases, and
Spycatcher/Spytag systems (Reddington & Howarth, 2015), realize the
formidable possibilities of protein composability and interfacing with a
variety of materials (Rashidian et al., 2013).
With this armamentarium of possibilities, PTM-enzymes represent a
treasure trove for chemical biologists seeking to leverage them for
fundamental and applied research. From a biomedical and drug discovery
point of view, molecules and other modalities that can control these
enzymes can lead to therapeutics and diagnostics reagents. From a
chemical biology and biotechnology point of view, there remains a need
to discover new PTM-enzymes and improve the properties of existing ones
for specific applications. In this vein, high-throughput protein
engineering and screening platforms are essential to harness the
possibilities PTM-enzymes can offer fully. Specifically, high-throughput
platforms allow one to profile PTM-enzyme activities, identify their
physiological substrates, screen inhibitors against specific
PTM-enzymes, and use protein engineering to improve existing properties
or introduce novel activities.
Many proteomic (Johnson et al., 2023; Schilling & Overall, 2007) and
genetic platforms (Dyer & Weiss, 2022) are now available to study and
engineer PTM-enzymes. Mass spectrometry-based methods based on protein
labeling are essential to assigning PTM-enzyme cellular substrates and
have been primarily applied to proteases and kinases. Genetic platforms
typically introduce the PTM-enzyme(s) of interest in a heterologous host
and devise a screening or selection method to assay enzyme activities.
Notable examples in bacterial hosts include phage-assisted continuous
evolution (Blum et al., 2021) and an OmpT-linked bacterial surface
display platform to engineer protease specificity (Ramesh et al., 2019;
Varadarajan et al., 2009; Varadarajan et al., 2005), and an E.
coli platform wherein cell survival is linked to protease inhibition
(Lopez et al., 2019). Over the past two decades, yeast, particularlySaccharomyces cerevisiae , has been a prominent organism in
PTM-enzyme investigations, in addition to its contribution as a model of
human diseases involving aberrant PTMs. S. cerevisiae has
countless genetic tools for controlling gene and protein expression and
editing its genome. Furthermore, our ability to generate large DNA
libraries in this organism enables protein engineering and directed
evolution campaigns. Yeast offers distinct advantages over bacterial
systems for studying PTM enzymes. These include expressing active human
PTM-enzymes and building spatially and temporally controlled genetic and
protein circuits empowered by organelle compartmentalization. By taking
advantage of yeast’s many protein sequestration mechanisms, one can
build high-throughput screening and selection platforms to engineer and
profile the substrate specificity of PTM-enzymes and screen for their
inhibitors.
This review discusses the principles and latest applications of S.
cerevisiae organelle sequestration to study and engineer PTM-enzymes,
particularly proteases and protein ligases. These include methods that
modify yeast surface display and employ enzyme-catalyzed transcription
activation. Lastly, we discuss yeast endoplasmic reticulum (ER)
sequestration, one of the more powerful methods to engineer PTM-enzymes.
Where appropriate, we highlight the major differences between these
approaches, including how they can measure and control enzyme catalytic
efficiencies.
Yeast surface display repurposed for PTM-enzyme engineering.