Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains |
| |
Authors: | T Vallon S Ghanegaonkar O Vielhauer A Müller C Albermann G Sprenger M Reuss K Lemuth |
| |
Institution: | (1) Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany;(2) Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany |
| |
Abstract: | In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a
precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate
(FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ
extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl
diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression
vector.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
T. Vallon and S. Ghanegaonkar contributed equally to this work. |
| |
Keywords: | Isoprenoid diphosphate Quantification Geranylgeranyl diphosphate synthase Heterologous expression Chromosomal integration |
本文献已被 PubMed SpringerLink 等数据库收录! |
|