Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis
Date
2019-11-05
Journal Title
Journal ISSN
Volume Title
Publisher
BMC (part of Springer Nature)
Abstract
Background: Terpenoids 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.
Results: We 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.
Discussion: These 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.
Description
CITATION: Volke, D. C., et al. 2019. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microbial Cell Factories, 18:192, doi:10.1186/s12934-019-1235-5.
The original publication is available at https://microbialcellfactories.biomedcentral.com
The original publication is available at https://microbialcellfactories.biomedcentral.com
Keywords
Metabolic control analysis, Terpenoids, Microbiology, Isoprene
Citation
Volke, D. C., et al. 2019. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microbial Cell Factories, 18:192, doi:10.1186/s12934-019-1235-5