Differential glucose repression in common yeast strains in response to HXK2 deletion.

Under aerobic, high glucose conditions, Saccharomyces cerevisiae exhibits glucose repression and thus a predominantly fermentative metabolism. Here, we show that two commonly used prototrophic representatives of the CEN.PK and S288C strain families respond differently to deletion of the hexokinase 2 (HXK2) - a key player in glucose repression: In CEN.PK, growth rate collapses and derepression occurs on the physiological level, while the S288C descendant FY4 Deltahxk2 still grows like the parent strain and shows a fully repressed metabolism. A CEN.PK Deltahxk2 strain with a repaired adenylate cyclase gene CYR1 maintains repression but not growth rate. A comparison of the parent strain's physiology, metabolome, and proteome revealed higher metabolic rates, identical biomass, and byproduct yields, suggesting a lower Snf1 activity and a higher protein kinase A (PKA) activity in CEN.PK. This study highlights the importance of the genetic background in the processes of glucose signaling and regulation, contributes novel evidence on the overlap between the classical glucose repression pathway and the cAMP/PKA signaling pathway, and might have the potential to resolve some of the conflicting findings existing in the field.

High-throughput quantitative metabolomics: workflow for cultivation, quenching, and analysis of yeast in a multiwell format.

Metabolomics is a founding pillar of quantitative biology and a valuable tool for studying metabolism and its regulation. Here we present a workflow for metabolomics in microplate format which affords high-throughput and yet quantitative monitoring of primary metabolism in microorganisms and in particular yeast. First, the most critical step of rapid sampling was adapted to a multiplex format by using fritted 96-well plates for cultivation, which ensure fast sample transfer and permit us to use well-established quenching in cold solvents. Second, extensive optimization of large-volume injection on a GC/TOF instrument provided the sensitivity necessary for robust quantification of 30 primary metabolites in 0.6 mg of yeast biomass. The metabolome profiles of baker's yeast cultivated in fritted well plates or in shake flasks were equivalent. Standard deviations of measured metabolites were between 10% and 50% within one plate. As a proof of principle we compared the metabolome of wild-type Saccharomyces cerevisiae and the single-deletion mutant Delta sdh1, which were clearly distinguishable by a 10-fold increase of the intracellular succinate concentration in the mutant. The described workflow allows the production of large amounts of metabolome samples within a day, is compatible with virtually all liquid extraction protocols, and paves the road to quantitative screens.

Cross-platform comparison of methods for quantitative metabolomics of primary metabolism.

Quantitative metabolomics is under intense development, and no commonly accepted standard analytical technique has emerged, yet. The employed analytical methods were mostly chosen based on educated guesses. So far, there has been no systematic cross-platform comparison of different separation and detection methods for quantitative metabolomics. Generally, the chromatographic separation of metabolites followed by their selective detection in a mass spectrometer (MS) is the most promising approach in terms of sensitivity and separation power. Using a defined mixture of 91 metabolites (covering glycolysis, pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, redox metabolism, amino acids, and nucleotides), we compared six separation methods designed for the analysis of these mostly very polar primary metabolites, two methods each for gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). For analyses on a single platform, LC provides the best combination of both versatility and robustness. If a second platform can be used, it is best complemented by GC. Only liquid-phase separation systems can handle large polar metabolites, such as those containing multiple phosphate groups. As assessed by supplementing the defined mixture with (13)C-labeled yeast extracts, matrix effects are a common phenomenon on all platforms. Therefore, suitable internal standards, such as (13)C-labeled biomass extracts, are mandatory for quantitative metabolomics with any methods.

The integrated response of primary metabolites to gene deletions and the environment.

Intracellular metabolites arise from the molecular integration of genomic and environmental factors that jointly determine metabolic activity. However, it is not clear how the interplay of genotype, nutrients, growth, and fluxes affect metabolite concentrations globally. Here we used quantitative metabolomics to assess the combined effect of environment and genotype on the metabolite composition of a yeast cell. We analyzed a panel of 34 yeast single-enzyme knockout mutants grown on three archetypical carbon sources, generating a dataset of 400 unique metabolome samples. The different carbon sources globally affected the concentrations of intermediates, both directly, by changing the thermodynamic potentials (Δ(r)G) as a result of the substrate influx, and indirectly, by cellular regulation. In contrast, enzyme deletion elicited only local accumulation of the metabolic substrate immediately upstream of the lesion. Key biosynthetic precursors and cofactors were generally robust under all tested perturbations in spite of changes in fluxes and growth rate.