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

• Published in: Applied microbiology and biotechnology Vol. 81; no. 1; pp. 175 - 182
• Main Authors: Vallon, T, Ghanegaonkar, S, Vielhauer, O, Müller, A, Albermann, C, Sprenger, G, Reuss, M, Lemuth, K
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.11.2008; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: Alcohols - metabolism; Biological and medical sciences; Biomass; Bioreactors - microbiology; Biosynthesis; Biotechnology; Carotenoids; Chromosomal integration; Chromosomes; Cloning; E coli; Enzymes; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; Gas chromatography; Gene Expression; Genetic Engineering; Genetic recombination; Geranylgeranyl diphosphate synthase; Heterologous expression; Isoprenoid diphosphate; Life Sciences; Mass spectrometry; Methods; Microbial Genetics and Genomics; Microbiology; Plasmids; Polyisoprenyl Phosphates - analysis; Polyisoprenyl Phosphates - isolation & purification; Polyisoprenyl Phosphates - metabolism; Quantitative analysis; Studies; Isoprenoid diphosphate; Quantification; Geranylgeranyl diphosphate synthase; Chromosomal integration; Heterologous expression; Enzyme; Transferases; Escherichia coli; Gene expression; Isoprenoid; Farnesyltranstransferase; Bacteria; Enterobacteriaceae; Quantitative analysis
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-008-1707-8

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.