Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli

Brett A. Boghigian, Daniel Salas, Parayil Kumaran Ajikumar, Gregory Stephanopoulos, Blaine A. Pfeifer

Taxa-4(5),11(12)-diene is the first dedicated intermediate in the metabolic pathway responsible for synthesizing the anticancer compound Taxol. In this study, the heterologous production of taxadiene was established in and analyzed between K- and B-derived Escherichia coli strains. First, recombinant parameters associated with precursor metabolism (the upstream methylerythritol phosphate (MEP) pathway) and taxadiene biosynthesis (the downstream pathway) were varied to probe the effect different promoters and cellular backgrounds have on taxadiene production. Specifically, upstream MEP pathway genes responsible for the taxadiene precursors, dimethylallyl diphosphate and isopentenyl diphosphate, were tested with an inducible T7 promoter system within K and B E. coli strains. Whereas, inducible T7, Trc, and T5 promoters were tested with the plasmid-borne geranylgeranyl diphosphate synthase and taxadiene synthase genes responsible for the downstream pathway. The K-derivative produced taxadiene roughly 2.5-fold higher than the B-derivative. A transcriptomics study revealed significant differences in pyruvate metabolism between the K and B strains, providing insight into the differences observed in taxadiene biosynthesis and targets for future metabolic engineering efforts. Next, the effect of temperature on cell growth and taxadiene production was analyzed in these two strains, revealing similar phenotypes between the two with 22°C as the optimal production temperature. Lastly, the effect of indole on cell growth was investigated between the two strains, showing that the K-derivative demonstrated greater growth inhibition compared to the B-derivative.