Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps—from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

Microbial superhost strains should provide an ideal platform for the efficient homologous or heterologous phenotypic expression of biosynthetic gene clusters (BGCs) of new and novel bioactive molecules. Our aim in the current study was to perform a comparative study at the bioprocess and metabolite levels of the previously designed superhost strain Streptomyces coelicolor M1152 and its derivative strain S. coelicolor M1581 heterologously expressing chloramphenicol BGC. Parent strain M1152 was characterized by a higher specific growth rate, specific CO₂ evolution rate, and a higher specific l‐glutamate consumption rate as compared with M1581. Intracellular primary central metabolites (nucleoside/sugar phosphates, amino acids, organic acids, and CoAs) were quantified using four targeted LC‐MS/MS‐based methods. The metabolite pathways in the nonantibiotic producing S. coelicolor host strain were flooded with carbon from both carbon sources, whereas in antibiotic‐producing strain, the carbon of l‐glutamate seems to be draining out through excreting synthesized antibiotic. The ¹³C‐isotope‐labeling experiments revealed the bidirectionality in the glycolytic pathway and reversibility in the non‐oxidative part of PPP even with continuous uptake of d‐glucose. The change in the primary metabolites due to the insertion of BGC disclosed a clear linkage between the primary and secondary metabolites.

Objective: The objective of this study was to investigate the variation of NAD and CoA metabolite pools in Saccharomyces cerevisiae cultivated under various cultivation conditions. This study complements a previous report on glycolytic, pentose phosphate pathway, tricarboxylic acid cycle, amino acids, and deoxy-/nucleoside phosphate pools determined under the same cultivation conditions. Results: S. cerevisiae pellets from batch (four carbohydrate sources) and chemostat (carbon-, nitrogen-, phosphate—limited and a range of dilution rates) bioreactor cultivations were extracted and analyzed with two recently established absolute quantitative liquid chromatography mass spectrometry (LC–MS/MS) methods for NAD and CoA metabolites. Both methods apply ¹³C internal standard dilution strategy for the enhanced analytical accuracy and precision. Individual metabolite pools were relatively constant for the different growth rates within the same mode of cultivation, but large differences were observed among some of the modes, i.e. NAD metabolites were 10 to 100-fold lower in nitrogen limited chemostats compared to the other modes, and phosphate limited chemostats were characterized with much lower CoA metabolite pools. The results complement the previous results and together provide a comprehensive insight into primary metabolite pools variations at a large range in growth and carbon source consumption rates.