Microscale Quantitative Analysis of Polyhydroxybutyrate in Prokaryotes Using IDMS

Metabolites
Mariana Itzel Velasco AlvarezS Aljoscha Wahl

Abstract

Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled (13)C-cell suspension, of an E. coli PHB producing strain, grown on U-(13)C-glucose as C-source. This internal (13)C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps.

References

Apr 7, 1999·Journal of Microbiological Methods·R P HesselmannA J Zehnder
Feb 28, 2001·Fresenius' Journal of Analytical Chemistry·D ElhottováH Santrůcková
Dec 13, 2005·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·James KacmarFriedrich Srienc
Jun 20, 2006·Mass Spectrometry Reviews·W M A NiessenR Andreoli
Dec 17, 2008·Analytical Biochemistry·Hilal Taymaz-NikerelWalter M van Gulik
Apr 9, 2014·Trends in Biotechnology·Stephan Noack, Wolfgang Wiechert
Sep 3, 2014·Methods in Molecular Biology·S Aljoscha WahlWalter M van Gulik
Oct 20, 2015·Biotechnology and Bioengineering·Sebastian NiedenführS Aljoscha Wahl

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Citations

Jul 28, 2020·The Science of the Total Environment·Jakub FojtJiří Kučerík
Jan 6, 2022·Applied Microbiology and Biotechnology·Chris M VermeerRobbert Kleerebezem

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Software Mentioned

Mass Hunter
Mass Hunter Quantitative Analysis software for MS

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