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

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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.

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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.

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Authors and Affiliations

  1. Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    T. Vallon, O. Vielhauer, A. Müller, M. Reuss & K. Lemuth

  2. Institute of Microbiology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    S. Ghanegaonkar, C. Albermann & G. Sprenger

Authors
  1. T. Vallon
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  2. S. Ghanegaonkar
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  3. O. Vielhauer
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  4. A. Müller
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  5. C. Albermann
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  6. G. Sprenger
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  7. M. Reuss
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  8. K. Lemuth
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Corresponding authors

Correspondence to C. Albermann or K. Lemuth.

Additional information

T. Vallon and S. Ghanegaonkar contributed equally to this work.

Electronic supplementary material

Fig. S1

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

Fig. S2

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

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)

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)

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)

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)

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Vallon, T., Ghanegaonkar, S., Vielhauer, O. et al. Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains. Appl Microbiol Biotechnol 81, 175–182 (2008). https://doi.org/10.1007/s00253-008-1707-8

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