Abstract
Isoprenoids are a highly diverse group of natural products with broad application as high value chemicals and advanced biofuels. They are synthesized using two primary building blocks, namely, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are generated via the mevalonate (MVA) or deoxy-d-xylulose-5-phosphate (DXP) pathways. Isoprenoid biosynthetic pathways are prevalent in eukaryotes, archaea, and bacteria. Measurement of isoprenoid intermediates via standard liquid chromatography–mass spectrometry (LC-MS) protocols is generally challenging because of the hydrophilicity and complex physicochemical properties of the molecules. In addition, there is currently no reliable analytical method that can simultaneously measure metabolic intermediates from MVA and DXP pathways, including the prenyl diphosphates. Therefore, we describe a robust hydrophilic interaction liquid chromatography time-of-flight mass spectrometry (HILIC-TOF-MS) method for analyzing isoprenoid intermediates from metabolically engineered Escherichia coli strains.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kirby J, Keasling JD (2009) Biosynthesis of plant isoprenoids: perspectives for microbial engineering. Annu Rev Plant Biol 60:335–355
Pitera DJ, Paddon CJ, Newman JD et al (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng 9:193–207
Gilibili RR, Kandaswamy M, Sharma K et al (2011) Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues. Biomed Chromatogr 25:1352–1359
Hayashi O, Satoh K (2014) Determination of acetyl-CoA and malonyl-CoA in germinating rice seeds using the LC-MS/MS technique. Biosci Biotechnol Biochem 70:2676–2681
Bartek T, Blombach B, Lang S et al (2011) Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum. Appl Environ Microbiol 77:6644–6652
Paczia N, Nilgen A, Lehmann T et al (2012) Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms. Microb Cell Factories 11:122
Kato H, Izumi Y, Hasunuma T et al (2012) Widely targeted metabolic profiling analysis of yeast central metabolites. J Biosci Bioeng 113:665–673
Fujii H, Koyama T, Ogura K (1982) Efficient enzymatic hydrolysis of polyprenyl pyrophosphates. Biochim Biophys Acta 712:716–718
Martin VJJ, Pitera DJ, Withers ST et al (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802
Henneman L, van CAG, Denis SW et al (2008) Detection of nonsterol isoprenoids by HPLC-MS/MS. Anal Biochem 383:18–24
Henneman L, van CAG, Kulik W et al (2011) Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS. Biochim Biophys Acta 1811:227–233
Zou R, Zhou K, Stephanopoulos G et al (2013) Combinatorial engineering of 1-deoxy-d-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA) method. PLoS One 8:e79557
Zhang D, Poulter CD (1993) Analaysis and purification of phosphorylated isoprenoids by reversed-phase HPLC. Anal Biochem 213:356–361
Li Z, Sharkey TD (2013) Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves. Plant Cell Environ 36:429–437
Zhou K, Zou R, Stephanopoulos G et al (2012) Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production. PLoS One 7:e47513
Bongers M, Chrysanthopoulos PK, Behrendorff JBYH et al (2015) Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite. Microb Cell Factories 14:193
Nowroozi FF, Baidoo EEK, Ermakov S et al (2014) Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly. Appl Microbiol Biotechnol 98:1567–1581
Acknowledgments
The authors would like to acknowledge that this work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Baidoo, E.E.K., Wang, G., Joshua, C.J., Benites, V.T., Keasling, J.D. (2019). Liquid Chromatography and Mass Spectrometry Analysis of Isoprenoid Intermediates in Escherichia coli. In: Baidoo, E. (eds) Microbial Metabolomics. Methods in Molecular Biology, vol 1859. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8757-3_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8757-3_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8756-6
Online ISBN: 978-1-4939-8757-3
eBook Packages: Springer Protocols