Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Brooks, Rani; Morici, Lisa; Sandoval, Nicholas

doi:10.1021/acssynbio.3c00239

Cited by 3 publications

(1 citation statement)

References 70 publications

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“…This transformative approach empowers researchers to finely craft phages with enhanced attributes, including refined host specificity, bolstered stability, and amplified therapeutic potential. Especially, the production of engineered phages can be strengthened by the use of cell-free phage synthesis technology [ 209 , 210 ].…”

Section: Future Perspectivesmentioning

confidence: 99%

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era

Jo,

Kwon,

Kim

et al. 2023

Microorganisms

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Amid the escalating challenges of antibiotic resistance, bacterial infections have emerged as a global threat. Bacteriophages (phages), viral entities capable of selectively infecting bacteria, are gaining momentum as promising alternatives to traditional antibiotics. Their distinctive attributes, including host specificity, inherent self-amplification, and potential synergy with antibiotics, render them compelling candidates. Phage engineering, a burgeoning discipline, involves the strategic modification of bacteriophages to enhance their therapeutic potential and broaden their applications. The integration of CRISPR-Cas systems facilitates precise genetic modifications, enabling phages to serve as carriers of functional genes/proteins, thereby enhancing diagnostics, drug delivery, and therapy. Phage engineering holds promise in transforming precision medicine, addressing antibiotic resistance, and advancing diverse applications. Emphasizing the profound therapeutic potential of phages, this review underscores their pivotal role in combatting bacterial diseases and highlights their significance in the post-antibiotic era.

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Section: Future Perspectivesmentioning

confidence: 99%

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era

Jo,

Kwon,

Kim

et al. 2023

Microorganisms

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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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Between Centralization and Fragmentation: The Past, Present, and Future of Phage Collections

Resch,

Brives,

Debarbieux

et al. 2024

PHAGE

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Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Cited by 3 publications

References 70 publications

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era

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

Between Centralization and Fragmentation: The Past, Present, and Future of Phage Collections

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