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Applied Microbiology and Biotechnology

, Volume 81, Issue 1, pp 175–182 | Cite as

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

  • T. Vallon
  • S. Ghanegaonkar
  • O. Vielhauer
  • A. Müller
  • C. Albermann
  • G. Sprenger
  • M. Reuss
  • K. Lemuth
Methods

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.

Keywords

Isoprenoid diphosphate Quantification Geranylgeranyl diphosphate synthase Heterologous expression Chromosomal integration 

Notes

Acknowledgements

The authors would like to thank Tom Schumacher for the determination of the glucose concentrations. This study was supported by the “Deutsche Forschungsgemeinschaft” (through collaborative research center SFB 706, TP B3) and the “Ministerium für Wissenschaft, Forschung und Kunst” of the state of Baden-Württemberg, Germany.

Supplementary material

253_2008_1707_Fig1_ESM.gif (24 kb)
Fig. S1

Mass spectrum of farnesol (FOH) peak at 10.3 min (GIF 23.7 KB)

253_2008_1707_Fig1_ESM.tiff.
High resolution image file (TIF 1.21 MB)
253_2008_1707_Fig2_ESM.gif (26 kb)
Fig. S2

Mass spectrum of geranylgeraniol (GGOH) peak at 15.0 min (GIF 25.8 KB)

253_2008_1707_Fig2_ESM.tiff.
High resolution image file (TIF 1.21 MB)
253_2008_1707_Fig3_ESM.gif (26 kb)
Fig. S3

Concentration of farnesol recovered from various concentrations of FPP after resuspension of evaporated standard solutions in dissolving solution and digestion with alkaline phosphatase. y = 1.1858x + 0.9242, R 2 = 0.9978 (GIF 26 KB)

253_2008_1707_Fig3_ESM.tiff.
High resolution image file (TIF 1.30 MB)
253_2008_1707_Fig4_ESM.gif (28 kb)
Fig. S4

Concentration of geranylgeraniol recovered from various concentrations of GGPP after resuspension of evaporated standard solutions in dissolving solution and digestion with alkaline phosphatase. y = 0.925x + 1.6084, R 2 = 0.9983 (GIF 28 KB)

253_2008_1707_Fig4_ESM.gif (28 kb)
High resolution image file (TIF 1.27 MB)
253_2008_1707_Fig5_ESM.gif (29 kb)
Fig. S5

FPP and GGPP were isolated from E. coli LJ110 after 10 h batch cultivation as described in the “Material and methods” section. Shown is the GC chromatogram of the hexane extract (GIF 29.4 KB)

253_2008_1707_Fig5_ESM.tiff.
High resolution image file (TIF 1.36 MB)
253_2008_1707_Fig6_ESM.gif (26 kb)
Fig. S6

FPP and GGPP were isolated from E. coli LJ110 malEG::Ptac-crtE after 30 h batch cultivation as described in the “Material and methods” section. Shown is the GC chromatogram of the hexane extract (GIF 26.1 KB)

253_2008_1707_Fig6_ESM.tiff.
High resolution image file (TIF 1.34 MB)
253_2008_1707_Fig7_ESM.gif (27 kb)
Fig. S7

FPP and GGPP were isolated from E. coli LJ110 with pCAS30 after 30 h batch cultivation as described in the “Material and methods” section. Shown is the GC chromatogram of the hexane extract (GIF 26.8 KB)

253_2008_1707_Fig7_ESM.tiff.
High resolution image file (TIF 1.34 MB)

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • T. Vallon
    • 1
  • S. Ghanegaonkar
    • 2
  • O. Vielhauer
    • 1
  • A. Müller
    • 1
  • C. Albermann
    • 2
  • G. Sprenger
    • 2
  • M. Reuss
    • 1
  • K. Lemuth
    • 1
  1. 1.Institute of Biochemical EngineeringUniversity of StuttgartStuttgartGermany
  2. 2.Institute of MicrobiologyUniversity of StuttgartStuttgartGermany

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