Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures

Abstract

The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

“…So far most authors considered the presence of metabolites of glycolysis, TCA cycle or PPP in the supernatant negligible, because the levels were below the detection limit of their analysis methods (Schaub et al, 2006 andHiller et al, 2007) However, this clearly does not agree with the present findings. Due to the sensitivity of the applied LC-ESI-ID-MS/MS analysis method metabolite concentrations well below 1 μM can be quantified.…”

“…They suggested using of cold methanol because of its simplicity and the fact that it allowed more components to be obtained. Hiller et al, 2007 claimed that usage of buffered hot water (30 mM TEA, pH 7.5, 95 °C) results in more reliable metabolite extraction compared to buffered ethanol, unbuffered hot water or perchloric acid for E. coli.…”

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

“…Even though sample preparation and extraction protocols were not the focus of this optimization, obtained results were consistent with previously published endogenous pool concentrations in E. coli under similar cultivation conditions. Notably, intracellular pool sizes are known to be significant depending on strain context, cultivation strategy, sample preparation, and assumed or investigated aqueous intracellular volumes [37][38][39][40]. Nevertheless, the intracellular metabolite concentrations of L-alanine and L-lysine are in good agreement with the reported measurements of Bennett and coworkers and Yuan and coworkers [38,39].…”

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry

“…When tested on E. coli and Saccharomyces cerevisiae and compared to other extraction methods (boiling ethanol, perchloric acid, or potassium hydroxide), the recoveries of intracellular metabolites after hot water extraction were excellent (Hans et al, 2001). Indeed, hot water was shown to extract efficiently phosphorylated metabolites such as nucleotides (Lundin and Thore, 1975) and intermediaries of glycolysis (Hiller et al, 2007). The recoveries that we obtained after hot water extraction were similar to those obtained by extraction with aqueous formic acid saturated with n-butanol on tobacco (Nicotiana tabacum) suspension cell cultures and cultured pollen tubes (Schlü pmann et al, 1994) and ice-cold methanol/chloroform (3:7, v/v) performed on Arabidopsis rosettes (Arrivault et al, 2009), and twice as high as the methanol/chloroform/formic acid/water (12:5:1:2, v/v/v/v) carried out on tobacco leaves (Cruz et al, 2008).…”