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Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...

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Profiling Expression Strategies for a Type III Polyketide Synthase in a Lysate-Based, Cell-free System.

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Cell-free expression (CFE) rapidly prototypes biosynthetic gene cluster (BGC) refactoring for improved natural product discovery. Codon harmonization in CFE enhances enzyme production more than traditional methods, accelerating research.

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Area of Science:

  • Microbiology
  • Biochemistry
  • Synthetic Biology

Background:

  • Expressing biosynthetic gene clusters (BGCs) from diverse bacteria like Actinobacteria in Escherichia coli presents challenges due to differences in DNA GC content, codon usage, and protein folding environments.
  • These challenges lead to unpredictable issues in protein expression and folding, significantly delaying the biochemical characterization of novel natural products.
  • Current BGC refactoring strategies for soluble, active enzyme expression in tractable hosts are often time-consuming and rely on trial-and-error.

Approach:

  • This study utilizes cell-free expression (CFE) as a platform for enzyme production and a testbed for rapid prototyping of refactoring techniques.
  • A type III polyketide synthase, RppA from Streptomyces griseus, was used as a reporter to investigate BGC refactoring, specifically focusing on improving flaviolin production.
  • The researchers synergistically tuned promoter and codon usage in CFE to optimize RppA expression and production of the natural product flaviolin.

Key Points:

  • Cell-free expression (CFE) serves as an effective platform for producing enzymes that are difficult to express in vivo.
  • CFE enables rapid prototyping and screening of refactoring strategies, including promoter and codon usage adjustments, before cellular implementation.
  • Codon harmonization demonstrated superior improvement in natural product synthesis compared to traditional codon optimization methods in both cell-free and cellular systems.

Conclusions:

  • Refactoring promoters and coding sequences using CFE is a valuable strategy for rapidly screening and achieving catalytically functional production of enzymes from BGCs.
  • The study highlights the coordination between CFE and in vivo expression, validating CFE as a powerful tool for accelerating natural product research.
  • Optimized CFE protocols can significantly de-risk and expedite the discovery and production of valuable natural products from microbial sources.