Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production
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Zhou-2012-Metabolite profiling.pdf
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Author(s)
Zhou, Kang
Zou, Ruiyang
Stephanopoulos, Gregory
Too, Heng-Phon
Date Issued
November 2012
Journal
PLoS ONE
Publisher
Public Library of Science
Citation
Zhou, Kang et al. “Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production.” Ed. Vasu D. Appanna. PLoS ONE 7.11 (2012): e47513.
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Final published version
Abstract
Isoprenoids are natural products that are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway. Metabolic engineering of microbes has enabled overproduction of various isoprenoid products from the DXP pathway including lycopene, artemisinic acid, taxadiene and levopimaradiene. To date, there is no method to accurately measure all the DXP metabolic intermediates simultaneously so as to enable the identification of potential flux limiting steps. In this study, a solid phase extraction coupled with ultra performance liquid chromatography mass spectrometry (SPE UPLC-MS) method was developed. This method was used to measure the DXP intermediates in genetically engineered E. coli. Unexpectedly, methylerythritol cyclodiphosphate (MEC) was found to efflux when certain enzymes of the pathway were over-expressed, demonstrating the existence of a novel competing pathway branch in the DXP metabolism. Guided by these findings, ispG was overexpressed and was found to effectively reduce the efflux of MEC inside the cells, resulting in a significant increase in downstream isoprenoid production. This study demonstrated the necessity to quantify metabolites enabling the identification of a hitherto unrecognized pathway and provided useful insights into rational design in metabolic engineering.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Singapore-MIT Alliance in Research and Technology (SMART)
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DOI of Published Version
https://doi.org/10.1371/journal.pone.0047513
