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

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.