Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production
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 o...
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Public Library of Science
2013
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| Online Access: | http://hdl.handle.net/1721.1/76647 https://orcid.org/0000-0001-6909-4568 |
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| author | Zhou, Kang Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Zhou, Kang Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon |
| author_sort | Zhou, Kang |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T10:17:05Z |
| format | Article |
| id | mit-1721.1/76647 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:17:05Z |
| publishDate | 2013 |
| publisher | Public Library of Science |
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| spelling | mit-1721.1/766472022-09-26T16:58:14Z Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production Zhou, Kang Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon Massachusetts Institute of Technology. Department of Chemical Engineering Singapore-MIT Alliance in Research and Technology (SMART) Zhou, Kang Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon 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. Singapore-MIT Alliance 2013-01-30T15:31:44Z 2013-01-30T15:31:44Z 2012-11 2012-05 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/76647 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. https://orcid.org/0000-0001-6909-4568 en_US http://dx.doi.org/10.1371/journal.pone.0047513 PLoS ONE Creative Commons Attribution http://creativecommons.org/licenses/by/2.5/ application/pdf Public Library of Science PLoS |
| spellingShingle | Zhou, Kang Zou, Ruiyang Stephanopoulos, Gregory Too, Heng-Phon Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production |
| title | Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production |
| title_full | Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production |
| title_fullStr | Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production |
| title_full_unstemmed | Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production |
| title_short | Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production |
| title_sort | metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production |
| url | http://hdl.handle.net/1721.1/76647 https://orcid.org/0000-0001-6909-4568 |
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