Nonlinear dependency of intracellular fluxes on growth rate in miniaturized continuous cultures of Escherichia coli.
A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic 13C analysis of intracellular fluxes over a broad range of growth rates in glucose-limited Escherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h(-1). During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO2. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h(-1) was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.
Nanchen
Annik
A
Institute of Molecular Systems Biology, ETH Zürich, CH-8093 Zürich, Switzerland.
Schicker
Alexander
A
Sauer
Uwe
U
D000332:Aerobiosis; D001431:Bacteriological Techniques / Q000295:instrumentation; D002952:Citric Acid Cycle; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D017711:Nonlinear Dynamics; D062907:Oxaloacetic Acid / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism
D006038:Glyoxylates; D062907:Oxaloacetic Acid; D010728:Phosphoenolpyruvate; D005947:Glucose; C031150:glyoxylic acid