Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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| Evidence ID | Analyze ID | File | Description |
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