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Glucose Pulse (glucose + pulse)

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Life Sciences	100%

Selected Abstracts

Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiae

FEMS YEAST RESEARCH, Issue 2 2002
Jasper A. Diderich
Abstract In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate. [source]

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses,

BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009
Alvaro R. Lara
Abstract The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed-acid fermentation metabolism was studied in a two-compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini-plug-flow reactor (PFR: BioScope, 3.5,mL volume). Such a system can mimic the situation often encountered in large-scale, fed-batch bioreactors. The STR represented the zones of a large-scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen-poor and glucose-rich regions can occur in large-scale bioreactors. The response of E. coli to these large-scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D,=,0.1,h,1) into the mini-PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92,s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2,s to the glucose-rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2,s after of exposure of E. coli cells to the glucose and O2 gradients. This approach (STR-mini-PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large-scale bioreactors and for the design of modified strains with an improved behavior under large-scale conditions. Biotechnol. Bioeng. 2009; 104: 1153,1161. © 2009 Wiley Periodicals, Inc. [source]

Stimulation, Monitoring, and Analysis of Pathway Dynamics by Metabolic Profiling in the Aromatic Amino Acid Pathway

BIOTECHNOLOGY PROGRESS, Issue 6 2004
M. Oldiges
Using a concerted approach of biochemical standard preparation, analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and statistical data analysis, the metabolism dynamics in the aromatic amino acid pathway has been stimulated, monitored, and analyzed in different tyrosine-auxotrophic l -phenylalanine-producing Escherichiacoli strains. During the observation window from ,4 s (before) up to 27 s after the glucose pulse, the dynamics of the first five enzymatic reactions in the aromatic amino acid pathway was observed by measuring intracellular concentrations of 3-deoxy- d -arabino-heptulosonate 7-phosphate DAH(P), 3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate 3-phosphate (S3P), and shikimate (SHI), together with the pathway precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose phosphate pool as an alternative to the nondetectable erythrose 4-phosphate (E4P). Provided that a sufficient fortification of the carbon flux into the pathway of interest is ensured, respective metabolism dynamics can be observed. On the basis of the intracellular pool measurements, the standardized pool velocities were calculated, and a simple, data-driven criterion-called "pool efflux capacity" (PEC)-is derived. Despite its simplifying system description, the criterion managed to identify the well-known AroB limitation in the E. coli strain A (genotype ,( pheA tyrA aroF)/pJF119EH aroFfbrpheAfbramp) and it also succeeded to identify AroL and AroA (in strain B, genotype ,( pheA tyrA aroF)/pJF119EH aroFfbrpheAfbraroB amp) as promising metabolic engineering targets to alleviate respective flux control in subsequent l -Phe producing strains. Furthermore, using of a simple correlation analysis, the reconstruction of the metabolite sequence of the observed pathway was enabled. The results underline the necessity to extend the focus of glucose pulse experiments by studying not only the central metabolism but also anabolic pathways. [source]