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Metabolites 2017, 7(2), 19;

Microscale Quantitative Analysis of Polyhydroxybutyrate in Prokaryotes Using IDMS

Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2629 HZ Delft, The Netherlands
BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands
Current address: DSM Food Specialties B.V., Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands.
Author to whom correspondence should be addressed.
Academic Editors: Wolfgang Eisenreich and Peter Meikle
Received: 22 March 2017 / Revised: 13 May 2017 / Accepted: 15 May 2017 / Published: 17 May 2017
(This article belongs to the Special Issue Isotope Guided Metabolomics and Flux Analysis)
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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 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-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. View Full-Text
Keywords: polyhydroxybutyrate; 3-hydroxy butyrate; GC-MS; 13C-labeled internal standard; isotope dilution; Escherichia coli polyhydroxybutyrate; 3-hydroxy butyrate; GC-MS; 13C-labeled internal standard; isotope dilution; Escherichia coli

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Velasco Alvarez, M.I.; ten Pierick, A.; van Dam, P.T.N.; Maleki Seifar, R.; van Loosdrecht, M.C.M.; Wahl, S.A. Microscale Quantitative Analysis of Polyhydroxybutyrate in Prokaryotes Using IDMS. Metabolites 2017, 7, 19.

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