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Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system
Tien T Sword1, Jaime Lorenzo N Dinglasan2,3, Ghaeath S K Abbas1,4
1Department of Chemistry, University of Tennessee-Knoxville, Knoxville, TN, USA.
Scientific Reports
|June 5, 2024
Summary
Codon harmonization and refactoring gene expression strategies in cell-free systems improve natural product discovery. This method rapidly screens for functional enzymes from diverse bacteria, advancing cell-free expression (CFE) for natural product research.
Area of Science:
- Microbiology
- Synthetic Biology
- Biochemistry
Background:
- Expressing genes from metabolically diverse bacteria like Actinobacteria in Escherichia coli presents challenges due to differences in DNA GC content and codon usage.
- These challenges lead to unpredictable issues in protein expression and folding, hindering the biochemical characterization of novel natural products.
- Current methods for achieving soluble and active protein expression in tractable hosts are often time-consuming and involve extensive trial-and-error.
Purpose of the Study:
- To investigate biosynthetic gene cluster (BGC) refactoring techniques using a type III polyketide synthase, RppA, as a reporter.
- To assess the utility of cell-free expression (CFE) systems for rapidly prototyping expression parameters before cellular implementation.
- To explore the synergistic effects of promoter choice and codon usage on protein expression.
Main Methods:
- A library of constructs with varying promoters and RppA coding sequences was created to study promoter-codon usage synergies.
- Cell-free expression (CFE) systems were utilized to prototype refactoring strategies.
- In vivo experiments were conducted to compare CFE results with cellular expression outcomes.
Main Results:
- Codon harmonization demonstrated superior improvement in natural product synthesis compared to traditional codon optimization in both cell-free and cellular systems.
- The interplay between coding sequences and promoters significantly impacts protein expression, highlighting the need for synergistic consideration in CFE.
- Promoter strategy effectiveness varied between RppA and Green Fluorescent Protein (GFP), indicating protein-specific promoter requirements.
Conclusions:
- Refactoring promoters and/or coding sequences using CFE is an effective strategy for rapid screening of catalytically functional enzymes from BGCs.
- CFE serves as a valuable tool for accelerating natural product research by enabling efficient prototyping of expression strategies.
- The findings underscore the importance of considering synergistic effects of genetic elements and expression environments for optimizing protein production.
