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Engineering cell-free systems by chemoproteomic-assisted phenotypic screening
Zarina Levitskaya1, Zheng Ser2, Hiromi Koh2
1Protein and Peptide Engineering and Research Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR) 8A Biomedical Grove Singapore 138648 fghadessy@imcb.a-star.edu.sg.
RSC Chemical Biology
|April 5, 2024
Summary
Phenotypic screening identified compounds that boost protein production in cell-free systems. This method, using ATP kinase inhibitors, enhanced yields by optimizing energy pathways for heterologous protein expression.
Area of Science:
- Biotechnology
- Synthetic Biology
- Molecular Biology
Background:
- Cell-free protein synthesis (CFPS) is crucial for biological system engineering.
- Optimizing CFPS yields requires understanding and manipulating complex cellular processes.
Purpose of the Study:
- To apply phenotypic screening to enhance yeast lysate CFPS.
- To identify compounds and genetic modifications that increase heterologous protein production.
Main Methods:
- Utilized target-agnostic phenotypic screening to identify compounds enhancing protein production in yeast CFPS.
- Investigated ATP kinase inhibitors and their role in redirecting ATP flux.
- Employed chemoproteomic-guided strain engineering, including gene deletion (SSA1).
- Combined drug-mediated metabolic rewiring with template optimization for improved yields.
Main Results:
- Identified numerous compounds, primarily ATP kinase inhibitors, that significantly enhanced protein production.
- Demonstrated a 30% increase in protein yield by deleting SSA1, an ATP-consuming HSP70 chaperone component.
- Achieved record protein yields in yeast CFPS using a cost-effective glucose regeneration system.
Conclusions:
- Phenotypic screening is effective for deconvoluting cell-lysate complexity in CFPS.
- Target identification and exploitation of compound mechanisms (e.g., ATP flux redirection) are key to improving CFPS.
- This approach expands strategies for enhancing CFPS efficiency and cost-effectiveness.
