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Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis.
Microb Cell Fact. 2019 Nov 05; 18(1):192.MC

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.

Links

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Authors+Show Affiliations

Volke DC
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
Rohwer J
Laboratory for Molecular Systems Biology, Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa.
Fischer R
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, USA.
Jennewein S
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany. Stefan.Jennewein@ime.fraunhofer.de.

MeSH

ButadienesErythritolEscherichia coliHemiterpenesMetabolic EngineeringMevalonic AcidSugar PhosphatesTerpenes

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31690314

Citation

Volke, Daniel Christoph, et al. "Investigation of the Methylerythritol 4-phosphate Pathway for Microbial Terpenoid Production Through Metabolic Control Analysis." Microbial Cell Factories, vol. 18, no. 1, 2019, p. 192.
Volke DC, Rohwer J, Fischer R, et al. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microb Cell Fact. 2019;18(1):192.
Volke, D. C., Rohwer, J., Fischer, R., & Jennewein, S. (2019). Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microbial Cell Factories, 18(1), 192. https://doi.org/10.1186/s12934-019-1235-5
Volke DC, et al. Investigation of the Methylerythritol 4-phosphate Pathway for Microbial Terpenoid Production Through Metabolic Control Analysis. Microb Cell Fact. 2019 Nov 5;18(1):192. PubMed PMID: 31690314.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. AU - Volke,Daniel Christoph, AU - Rohwer,Johann, AU - Fischer,Rainer, AU - Jennewein,Stefan, Y1 - 2019/11/05/ PY - 2019/06/28/received PY - 2019/10/17/accepted PY - 2019/11/7/entrez PY - 2019/11/7/pubmed PY - 2020/2/6/medline KW - E. coli KW - Isoprene KW - MEP pathway KW - Metabolic control analysis KW - Recombineering SP - 192 EP - 192 JF - Microbial cell factories JO - Microb Cell Fact VL - 18 IS - 1 N2 - 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. SN - 1475-2859 UR - https://www.unboundmedicine.com/medline/citation/31690314/Investigation_of_the_methylerythritol_4_phosphate_pathway_for_microbial_terpenoid_production_through_metabolic_control_analysis_ DB - PRIME DP - Unbound Medicine ER -