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 13 C 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 CO 2 . 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.The flexible nature of metabolic networks is based on an extensive set of biochemical reactions whose activity is modulated by complex regulatory networks that operate at multiple levels. The core of this intricate network consists of about 100 central metabolic reactions that synthesize biosynthetic building blocks and regenerate cofactors from a wide variety of substrates. To understand the behavior and regulation of such networks, quantitative data on intracellular fluxes under different conditions and with defined mutants are required. A common problem with mutants, however, is their often dramatic differences in growth rates (2,11,50), such that detected flux differences may be nonspecific, growth rate-related phenomena (4, 29, 46).To ensure highly comparable conditions, continuous cultures are the method of choice because, unlike with batch cultures, a defined steady-state growth rate that equals the externally controlled dilution rate is maintained (17, 31). The labor and cost associated with continuous cultures in controlled bioreactors, however, preclude systematic experiments on any larger scale. For bioprocess development in batch and fed-batch cultures, parallel reactor systems are becoming available (for a recent review, see reference 49), and microtiter plate-based cultivation devices were successfully used for quantitative batch experiments (2, 6, 11, 21, 51). Small-scale continuous-culture devices, in contrast, are used mostly...

Although a whole arsenal of mechanisms are potentially involved in metabolic regulation, it is largely uncertain when, under which conditions, and to which extent a particular mechanism actually controls network fluxes and thus cellular physiology. Based on 13 C flux analysis of Escherichia coli mutants, we elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, CreB, CreC, Crp, Cya, Fnr, Hns, Mlc, OmpR, and UspA on aerobic glucose catabolism in glucose-limited chemostat cultures at a growth rate of 0.1 h ؊1 . The by far most relevant control mechanism was cyclic AMP (cAMP)-dependent catabolite repression as the inducer of the phosphoenolpyruvate (PEP)-glyoxylate cycle and thus low tricarboxylic acid cycle fluxes. While all other mutants and the reference E. coli strain exhibited high glyoxylate shunt and PEP carboxykinase fluxes, and thus high PEP-glyoxylate cycle flux, this cycle was essentially abolished in both the Crp and Cya mutants, which lack the cAMP-cAMP receptor protein complex. Most other mutations were phenotypically silent, and only the Cra and Hns mutants exhibited slightly altered flux distributions through PEP carboxykinase and the tricarboxylic acid cycle, respectively. The Cra effect on PEP carboxykinase was probably the consequence of a specific control mechanism, while the Hns effect appears to be unspecific. For central metabolism, the available data thus suggest that a single transcriptional regulation process exerts the dominant control under a given condition and this control is highly specific for a single pathway or cycle within the network.Some parts of metabolic networks are organism specific, but the core network is highly conserved. Almost all aerobic bacteria have a similar set of about 100 enzymes that catalyze the formation of biosynthetic building blocks, energy, and cofactors. This core network is ubiquitous because all specialized catabolic pathways finally merge into one or more of the common intermediates. Obviously, not all core reactions are simultaneously active and the evolved regulatory structure of an organism ensures appropriate and flexible activity of the various enzymes under the conditions normally encountered. One key regulatory task is to direct carbon fluxes such that all of the necessary biomass components are synthesized at the appropriate stoichiometry and rate from a wide range of substrates.Transcriptional regulation is generally considered the main microbial control mechanism, and a complicated network of global and specific transcription factors could potentially manage this distribution of fluxes (31). Such transcriptional control of metabolic activity is firmly established for the degradation and biosynthesis branches of the network. A typical example is aromatic amino acid biosynthesis with fine tuning of flux into the various branches by allosteric feedback inhibition of key enzymes but transcriptional regulation as the general control mechanism for absence or presence of the pathway (41). The situation is much less clear, ...

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