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

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 frequently results in a myriad of unpredictable issues with 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, such as BGC refactoring, can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as 1) a valuable expression platform for enzymes that are challenging to synthesize in vivo, and as 2) a testbed for rapid prototyping that can improve cellular expression. 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 synergistically tune promoter and codon usage to improve flaviolin production from cell-free expressed RppA. We then 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. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs. By showing the coordinators between CFE versus in vivo expression, this work advances CFE as a tool for natural product research.