Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System
Jaime Lorenzo N. Dinglasan, Tien T. Sword, J. William Barker, Mitchel J. Doktycz, and Constance B. Bailey
April 11, 2023, ACS Synthetic Biology; DOI: 10.1021/acssynbio.2c00658
Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein’s catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. The reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.
Dinglasan JLN, Sword TT, Barker JW, Doktycz MJ, and Bailey CB (2023). Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System. ACS Synthetic Biology, doi:10.1021/acssynbio.2c00658.