Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains
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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.
KeywordsIsoprenoid diphosphate Quantification Geranylgeranyl diphosphate synthase Heterologous expression Chromosomal integration
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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