Rational search of genetic design space for a heterologous terpene metabolic pathway in Streptomyces

Modern tools in DNA synthesis and assembly give genetic engineers control over the nucleotide-level design of complex, multi-gene systems. Systematic approaches to explore genetic design space and optimize the performance of genetic constructs are lacking. Here we explore the application of a five-level Plackett-Burman fractional factorial design to improve the titer of a heterologous terpene biosynthetic pathway in Streptomyces. A library of 125 engineered gene clusters encoding the production of diterpenoid ent-atiserenoic acid (eAA) via the methylerythritol phosphate pathway was constructed and introduced into Streptomyces albidoflavus J1047 for heterologous expression. The eAA production titer varied within the library by over two orders of magnitude and host strains showed unexpected and reproducible colony morphology phenotypes. Analysis of Plackett-Burman design identified expression of dxs, the gene encoding the first and the flux-controlling enzyme, having the strongest impact on eAA titer, but with a counter-intuitive negative correlation between dxs expression and eAA production. Finally, simulation modeling was performed to determine how several plausible sources of experimental error/noise and non-linearity impact the utility of Plackett-Burman analyses.