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.