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...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2012-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3487848?pdf=render |
| _version_ | 1819042767908634624 |
|---|---|
| author | Kang Zhou Ruiyang Zou Gregory Stephanopoulos Heng-Phon Too |
| author_facet | Kang Zhou Ruiyang Zou Gregory Stephanopoulos Heng-Phon Too |
| author_sort | Kang Zhou |
| collection | DOAJ |
| 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 | 2024-12-21T09:46:08Z |
| format | Article |
| id | doaj.art-c1dc866f46ab47ae86dbb97a082c919c |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T09:46:08Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-c1dc866f46ab47ae86dbb97a082c919c2022-12-21T19:08:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01711e4751310.1371/journal.pone.0047513Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production.Kang ZhouRuiyang ZouGregory StephanopoulosHeng-Phon TooIsoprenoids 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.http://europepmc.org/articles/PMC3487848?pdf=render |
| spellingShingle | Kang Zhou Ruiyang Zou Gregory Stephanopoulos Heng-Phon Too Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production. PLoS ONE |
| 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://europepmc.org/articles/PMC3487848?pdf=render |
| work_keys_str_mv | AT kangzhou metaboliteprofilingidentifiedmethylerythritolcyclodiphosphateeffluxasalimitingstepinmicrobialisoprenoidproduction AT ruiyangzou metaboliteprofilingidentifiedmethylerythritolcyclodiphosphateeffluxasalimitingstepinmicrobialisoprenoidproduction AT gregorystephanopoulos metaboliteprofilingidentifiedmethylerythritolcyclodiphosphateeffluxasalimitingstepinmicrobialisoprenoidproduction AT hengphontoo metaboliteprofilingidentifiedmethylerythritolcyclodiphosphateeffluxasalimitingstepinmicrobialisoprenoidproduction |