Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast.

BACKGROUND: Natural and industrial environments are dynamic with respect to substrate availability and other conditions like temperature and pH. Especially, metabolism is strongly affected by changes in the extracellular space. Here we study the dynamic flux of central carbon metabolism and storage carbohydrate metabolism under dynamic feast/famine conditions in Saccharomyces cerevisiae. RESULTS: The metabolic flux reacts fast and sensitive to cyclic perturbations in substrate availability. Compared to well-documented stimulus-response experiments using substrate pulses, different metabolic responses are observed. Especially, cells experiencing cyclic perturbations do not show a drop in ATP with the addition of glucose, but an immediate increase in energy charge. Although a high glycolytic flux of up to 5.4 mmol g DW-1  h-1 is observed, no overflow metabolites are detected. From famine to feast the glucose uptake rate increased from 170 to 4788 µmol g DW-1  h-1 in 24 s. Intracellularly, even more drastic changes were observed. Especially, the T6P synthesis rate increased more than 100-fold upon glucose addition. This response indicates that the storage metabolism is very sensitive to changes in glycolytic flux and counterbalances these rapid changes by diverting flux into large pools to prevent substrate accelerated death and potentially refill the central metabolism when substrates become scarce. Using 13C-tracer we found a dilution in the labeling of extracellular glucose, G6P, T6P and other metabolites, indicating an influx of unlabeled carbon. It is shown that glycogen and trehalose degradation via different routes could explain these observations. Based on the 13C labeling in average 15% of the carbon inflow is recycled via trehalose and glycogen. This average fraction is comparable to the steady-state turnover, but changes significantly during the cycle, indicating the relevance for dynamic regulation of the metabolic flux. CONCLUSIONS: Comparable to electric energy grids, metabolism seems to use storage units to buffer peaks and keep reserves to maintain a robust function. During the applied fast feast/famine conditions about 15% of the metabolized carbon were recycled in storage metabolism. Additionally, the resources were distributed different to steady-state conditions. Most remarkably is a fivefold increased flux towards PPP that generated a reversed flux of transaldolase and the F6P-producing transketolase reactions. Combined with slight changes in the biomass composition, the yield decrease of 5% can be explained.

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average half-life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

MIRACLE: mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites.

First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography-electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (mu)=0.05 h(-1) in a medium containing only naturally labeled (99% U-12C, 1% U-13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h approximately 80% of the measured glycolytic metabolites were U-13C-labeled. Surprisingly, during the next 3 h no significant increase of the U-13C-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-13C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-13C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-13C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-13C-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U-13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.

Kinetics of ferrous iron oxidation by Leptospirillum bacteria in continuous cultures.

The oxidation of ferrous iron by Leptospirillum bacteria was studied in a continuous culture in the dilution rate range 0.009-0.077 h-1 and could be described with a rate equation for competitive ferric iron inhibition kinetics in terms of the ferric/ferrous iron ratio in the solution. The ferrous iron oxidation in the continuous culture was followed by means of oxygen and carbon dioxide concentration analyses in reference air and off-gas. From these measurements the oxygen consumption rate, rO2, the carbon dioxide consumption rate, rCO2, the biomass concentration, Cx, and the biomass specific oxygen consumption rate, qO2, in the culture were determined. The ferrous iron concentration in the culture was below accurate levels to determine with the usual titrimetric method and was therefore derived from measuring the solution redox potential. The degree of reduction balance was used to check the theoretically expected relation between the rates of ferrous iron, -rFe2+, oxygen, -rO2, and biomass, rx. The maximum biomass yield and maintenance coefficient on oxygen are Yoxmax = 0.047 mol of C/mol of O2 and mo = 0.057 mol of O2/(mol of C.h). The maximum specific oxygen consumption rate, qO2,max = 1.7 mol of O2/(mol of C.h), the affinity coefficient, Ks/Ki = 0.0005 mol of Fe2+/mol of Fe3+, and the maximum specific growth rate, micromax = 0.069 h-1, Ks/Ki = 0.0004, were fitted from the measured data. For several dilution rates, off-line respiratory measurements with cell suspension from the continuous culture were carried out in dynamic BOM-Eh measurements. The dissolved oxygen and redox potential were measured simultaneously and monitored. The measured value of qO2,max varied between 2.3 and 1.7 mol/(mol of C.h). The value of Ks/Ki = 0.0007 was equal in all experiments. The measured values of qO2 in the continuous culture were well described with the kinetics determined in dynamic BOM-Eh measurements. It was concluded that dynamic BOM-Eh measurements are a convenient method to determine the kinetics of continuous culture grown Leptospirillum bacteria.

Determination of oxygen profiles in biocatalyst particles by means of a combined polarographic oxygen microsensor.

When studying the effect of immobilization of enzymes or whole cells on the conversion of substrate, more information is gained if measurements of substrate inside the biocatalyst particles are possible. With the methods used until now, only measurements outside the particle can be performed. In this article a method for measuring oxygen profiles in a biocatalyst particle under steady state conditions is described. The biocatalyst particle was made of agarose and contained the enzyme L-lactate 2-monooxygenase. This enzyme decarboxylates lactic acid to acetic acid in the presence of oxygen. The experiments were carried out in a flow chamber with the use of a micromanipulator and a stereomicroscope. The data were sampled by means of a computer. Four different profiles were measured using four different enzyme concentrations. The measured oxygen profiles were reproducible and the signal was very stable. It was also possible to measure the boundary layer around the particle. With the use of the oxygen microsensor, measurements in a biocatalyst particle could be performed accurately, giving way for model validation.