Abstract
BackgroundTerpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood.ResultsWe applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and isopentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a flux control coefficient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway flux) in the isoprene-producing strain and therefore exerted significant control over the flux though the MEP pathway. At higher dxs expression levels, the intracellular concentration of 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the flux towards isoprene. The higher intracellular concentrations of MEcPP led to the efflux of this intermediate into the growth medium.DiscussionThese findings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineering of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids.
Highlights
Terpenoids are of high interest as chemical building blocks and pharmaceuticals
Others have found indications for the intrinsic regulation of the methyl-d-erythritol 4-phosphate (MEP) pathway at the steps catalyzed by 1-deoxyd-xylulose-5-phosphate synthase (Dxs) and 1-deoxyd-xylulose-5-phosphate reductoisomerase (Dxr) [10], 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase (IspD) [11] or 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate synthase (IspF) [12]
Because IspS produces isoprene from dimethylallyl pyrophosphate (DMAPP) via the elimination of diphosphate (Fig. 1c), we speculated that the catalytic activity of Idi was too low to keep up with the consumption of DMAPP by IspS, which would result in low concentrations of DMAPP
Summary
Terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. The MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. All terpenoids originate from the isomeric precursors dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), both of which are produced via one of two natural metabolic routes: the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway [5]. The MVA pathway has been engineered to produce large quantities of terpenoids [6, 7] but attempts to engineer the MEP pathway have been less successful [8, 9]. The behavior of the MEP pathway has not been described in a precise and quantitative manner either in wild-type or engineered microbes
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