Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

Dinglasan, Jaime Lorenzo N.; Sword, Tien T.; Barker, J. William; Doktycz, Mitchel J.; Bailey, Constance B.

doi:10.1021/acssynbio.2c00658

Title: Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

Journal Article · Tue Apr 11 00:00:00 EDT 2023 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.2c00658· OSTI ID:1999071

Dinglasan, Jaime Lorenzo N. ^[1]; Sword, Tien T. ^[2]; Barker, J. William ^[2];

^[3];

^[2]

University of Tennessee, Knoxville, TN (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
University of Tennessee, Knoxville, TN (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

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. In conclusion, the reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)

Grant/Contract Number:: AC05-00OR22725; R15GM146192

OSTI ID:: 1999071

Journal Information:: ACS Synthetic Biology, Vol. 12, Issue 5; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Chemical reactions
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cell-free protein expression
nonribosomal peptide synthetase
natural products
blue pigment synthetase A
fluorescent labeling

Title: Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

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