Respirometric evaluation and modeling of glucose utilization by Escherichia coli under aerobic and mesophilic cultivation conditions.

The study presents a mechanistic model for the evaluation of glucose utilization by Escherichia coli under aerobic and mesophilic growth conditions. In the first step, the experimental data was derived from batch respirometric experiments conducted at 37 degrees C, using two different initial substrate to microorganism (S(0)/X(0)) ratios of 15.0 and 1.3 mgCOD/mgSS. Acetate generation, glycogen formation and oxygen uptake rate profile were monitored together with glucose uptake and biomass increase throughout the experiments. The oxygen uptake rate (OUR) exhibited a typical profile accounting for growth on glucose, acetate and glycogen. No acetate formation (overflow) was detected at low initial S(0)/X(0) ratio. In the second step, the effect of culture history developed under long-term growth limiting conditions on the kinetics of glucose utilization by the same culture was evaluated in a sequencing batch reactor (SBR). The system was operated at cyclic steady state with a constant mean cell residence time of 5 days. The kinetic response of E.coli culture was followed by similar measurements within a complete cycle. Model calibration for the SBR system showed that E. coli culture regulated its growth metabolism by decreasing the maximum growth rate (lower microH) together with an increase of substrate affinity (lower K(S)) as compared to uncontrolled growth conditions. The continuous low rate operation of SBR system induced a significant biochemical substrate storage capability as glycogen in parallel to growth, which persisted throughout the operation. The acetate overflow was observed again as an important mechanism to be accounted for in the evaluation of process kinetics.

Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids.

Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.

Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae.

Yeast vacuoles are highly dynamic and flexible organelles. In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for sample preparation that so far have been barely successful in yeast. Cells of leucine-limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase. To relieve leucine-limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid-based expression of a leucine marker gene. Such cultures reached more than two-fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.