Increasing diterpene yield with a modular metabolic engineering system in E. coli: Comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product.