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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-009-2219-x) contains supplementary material, which is available to authorized users.

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…S2). This finding was consistent with previous reports that enhancement in production of various metabolites requires only dxs and idi but not ispDF to be overexpressed or modulated (Jin and Stephanopoulos 2007;Morrone et al 2010;Sun et al 2014) and that the overexpression of ispDF operon even decreased isoprenoid production . The enzymes ispD and ispF were reported to be more soluble than the enzymes dxs and idi and may have higher activities (Zhou et al 2012); hence, the overexpression of ispDF may be unnecessary when the pathway was optimized, and further study is required to address this issue.…”

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

“…10 These strategies commonly focus on the mevalonic acid (MVA) or methyl-D-erythritol phosphate (MEP) pathways that are at the early stage of terpenoids biosynthesis. 6,7,11 However, the conservation and interactions of diterpenoids synthases attracted little attention.…”

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…An established Escherichia coli coexpression system (Morrone et al, 2010) was used to analyze the coupled activities of ZmKSL4 with (1) the ent-CPP synthase ZmAN2, (2) the rice syn-CPP synthase (OsCPS4; Xu et al, 2004), and (3) the grand fir (Abies grandis) abietadiene synthase variant D621A that produces CPP of normal [i.e. (+)] stereochemistry (Morrone et al, 2010). These three different combinations were selected, since ent-, syn-, and (+)-CPP represent the known stereochemical variations of class II diTPS products occurring naturally in many monocots (Peters, 2010;Schmelz et al, 2014).…”

“…An established Escherichia coli coexpression system (Morrone et al, 2010) was used to analyze the coupled activities of ZmKSL4 with (1) the ent-CPP synthase ZmAN2, (2) the rice syn-CPP synthase (OsCPS4; Xu et al, 2004), and (3) the grand fir (Abies grandis) abietadiene synthase variant D621A that produces CPP of normal [i.e. (+)] stereochemistry (Morrone et al, 2010).…”

“…The functional coexpression of enzymes was carried out using a previously described E. coli system engineered for enhanced diterpenoid production (Morrone et al, 2010;Kitaoka et al, 2015). For biochemical characterization of ZmKSL4, the N-terminally truncated gene in the expression vector pET28b(+) or pCOLA-DUET was cotransformed in E. coli BL21DE3-C41 cells (Lucigen) with a plasmid carrying a geranylgeranyl diphosphate synthase and constructs of class II diTPSs with different products: ZmAN2 forming ent-CPP (pGGeC), rice (Oryza sativa) CPS4 producing syn-CPP (pGGsC), or a variant of grand fir (Abies grandis) abietadiene synthase forming (+)-CPP (pGGnC; Morrone et al, 2010).…”