Dataset Information

Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production.

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.

SUBMITTER: Zhou K

PROVIDER: S-EPMC3487848 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production.

Zhou Kang K Zou Ruiyang R Stephanopoulos Gregory G Too Heng-Phon HP

PloS one 20121102 11

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 met ...[more]

PMID: 23133596

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Project description:High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade. Lycopene production is known to vary between wild-type Escherichia coli hosts, but the reasons behind this variation have never been fully elucidated.In an examination of six E. coli strains we observed that strains also differ in their capacity for increased lycopene production in response to metabolic engineering. A combination of genetic complementation, quantitative SWATH proteomics, and biochemical analysis in closely-related strains was used to examine the mechanistic reasons for variation in lycopene accumulation. This study revealed that rpoS, a gene previously identified in lycopene production association studies, exerts its effect on lycopene accumulation not through modulation of pathway flux, but through alteration of cellular oxidative status. Specifically, absence of rpoS results in increased accumulation of reactive oxygen species during late log and stationary phases. This change in cellular redox has no effect on isoprenoid pathway flux, despite the presence of oxygen-sensitive iron-sulphur cluster enzymes and the heavy redox requirements of the methylerythritol phosphate pathway. Instead, decreased cellular lycopene in the ?rpoS strain is caused by degradation of lycopene in the presence of excess reactive oxygen species.Our results demonstrate that lycopene is not a reliable indicator of isoprenoid pathway flux in the presence of oxidative stress, and suggest that caution should be exercised when using lycopene as a screening tool in genome-wide metabolic engineering studies. More extensive use of systems biology for strain analysis will help elucidate such unpredictable side-effects in metabolic engineering projects.

Dataset Information

Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production.

Publications

Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production.

Similar Datasets

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