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Leveraging yeast sequestration to study and engineer post-translational modification enzymes.
  • Carl Denard,
  • Samantha G. Martinusen
Carl Denard
University of Florida Department of Chemical Engineering

Corresponding Author:[email protected]

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Samantha G. Martinusen
University of Florida Department of Chemical Engineering
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Enzymes that catalyze post-translational modifications of peptides and proteins (PTM-enzymes) – proteases, protein ligases, oxidoreductases, kinases, and other transferases - are foundational to our understanding of health and disease and empower applications in chemical biology, synthetic biology, and biomedicine. To fully harness the potential of PTM-enzymes, there is a critical need to decipher their enzymatic and biological mechanisms, develop molecules that can probe and reprogram them, and endow them with improved and novel functions. These objectives are contingent upon implementation of high-throughput functional screens and selections that interrogate large sequence libraries to isolate desired PTM-enzyme properties. This review discusses the principles of S. cerevisiae organelle sequestration to study and engineer PTM-enzymes. These include methods that modify yeast surface display and employ enzyme-mediated transcription activation to evolve the activity and substrate specificity of proteases and protein ligases. We also present a detailed discussion of yeast endoplasmic reticulum (ER) sequestration for the first time. Where appropriate, we highlight the major features and limitations of different systems, specifically how they can measure and control enzyme catalytic efficiencies. Taken together, yeast-based high-throughput sequestration approaches significantly lower the barrier to understanding how PTM-enzymes function and how to reprogram them.
17 May 2023Submitted to Biotechnology and Bioengineering
17 May 2023Assigned to Editor
17 May 2023Submission Checks Completed
17 May 2023Review(s) Completed, Editorial Evaluation Pending
18 May 2023Reviewer(s) Assigned
23 Sep 2023Editorial Decision: Revise Major
02 Nov 20231st Revision Received
14 Nov 2023Submission Checks Completed
14 Nov 2023Assigned to Editor
14 Nov 2023Review(s) Completed, Editorial Evaluation Pending