What remains from living cells in bacterial lysate-based cell-free systems

Wagner, Lutz; Jules, Matthieu; Borkowski, Olivier

doi:10.1016/j.csbj.2023.05.025

Cited by 5 publications

(1 citation statement)

References 141 publications

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“…Cell-free expression (CFE) platforms employ either crude cell lysates (or extracts) or an in vitro transcription and translation (TX-TL) PURE system and can bypass limitations in secondary metabolite production observed in vivo expression systems 7 , 8 . The TX-TL PURE system or PURExpress employs the minimal number of recombinant elements required for transcription and translation while approaches using crude cell lysates are derived from intact living cells.…”

Section: Introductionmentioning

confidence: 99%

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Sword,

Dinglasan,

Abbas

et al. 2024

Sci Rep

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Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

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Section: Introductionmentioning

confidence: 99%

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Sword,

Dinglasan,

Abbas

et al. 2024

Sci Rep

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Nano-sized lysate of Lactiplantibacillus plantarum isolated from green tea leaves as a potential skin care ingredient

Myoung,

Choi,

Kim

et al. 2024

Biotechnol Bioproc E

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Synthetic Biology of Natural Products Engineering: Recent Advances Across the Discover–Design–Build–Test–Learn Cycle

Foldi,

Connolly,

Takano

et al. 2024

ACS Synth. Biol.

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Advances in genome engineering and associated technologies have reinvigorated natural products research. Here we highlight the latest developments in the field across the discover− design−build−test−learn cycle of bioengineering, from recent progress in computational tools for AI-supported genome mining, enzyme and pathway engineering, and compound identification to novel host systems and new techniques for improving production levels, and place these trends in the context of responsible research and innovation, emphasizing the importance of anticipatory analysis at the early stages of process development.

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What remains from living cells in bacterial lysate-based cell-free systems

Cited by 5 publications

References 141 publications

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Nano-sized lysate of Lactiplantibacillus plantarum isolated from green tea leaves as a potential skin care ingredient

Synthetic Biology of Natural Products Engineering: Recent Advances Across the Discover–Design–Build–Test–Learn Cycle

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