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.
- Albermann C, Ghanegaonkar S, Lemuth K, Vallon T, Reuss M, Armbruster W, Sprenger GA (2008) Biosynthesis of the Vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells. ChemBioChem (in press)Google Scholar
- Baba T, Muth J, Allen CM (1985) Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue. J Biol Chem 260:10467–10473Google Scholar
- Chiang CJ, Chen PT, Chao YP (2008) Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli. Biotechnol Bioeng. doi: 10.1002/bit.21976
- Fujisaki S, Nishino T, Katsuki H (1986) Isoprenoid synthesis in Escherichia coli. Separation and partial purification of four enzymes involved in the synthesis. J Biochem 99:1327–1337Google Scholar
- Kato J, Fujisaki S, Nakajima K, Nishimura Y, Sato M, Nakano A (1999) The Escherichia coli homologue of yeast RER2, a key enzyme of dolichol synthesis, is essential for carrier lipid formation in bacterial cell wall synthesis. J Bacteriol 181:2733–2738Google Scholar
- Keller RK (1996) Squalene synthase inhibition alters metabolism of nonsterols in rat liver. Biochim Biophys Acta 1303:169–179Google Scholar
- Lee PC, Mijts BN, Schmidt-Dannert C (2004) Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 65:538–546Google Scholar
- Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa Y, Nakamura K, Harashima K (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J Bacteriol 172:6704–6712Google Scholar
- Rohmer M, Knani M, Simonin P, Sutter B, Sahm H (1993) Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J 295(Pt 2):517–524Google Scholar