Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures ofEscherichia coli

Abstract

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...

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

“…The measured biomass yield in our chemostat cultivations was Y SX =3.2 CmolX/mol glucose which is very similar to published biomass yields of this E. coli strain under similar conditions (Pramanik and Keasling, 1997;Nanchen et al, 2006). From the last two columns of Table 2 it can be seen that the recoveries of carbon and redox were close to 100% for both the 4 L and the 0.5 L chemostats.…”

Functioning of a metabolic flux sensor in Escherichia coli

“…Indeed, when we experimentally perturbed the glycolytic flux through glucose-limited chemostat cultures (13) and used 13 Cmetabolic flux analysis (14,15) to quantify the flux through glycolysis, we found that the Cra activity-inferred from GFP-based transcriptional reporter plasmids (16) (Materials and Methods)-correlates with the glycolytic flux (Fig. 1B).…”

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

“…4 and 5). Typically, wild-type E. coli strains, including E. coli JM101, form acetate above critical glucose uptake rates of 3.4 to 5.5 mmol (g CDW) Ϫ1 h Ϫ1 at growth rates and temperatures of between 0.35 and 0.4 h Ϫ1 and 28 and 37°C, respectively (16,31,44). The presence of NADH oxidase increased the critical glucose uptake rate from 4.44 to 6.67 mmol (g CDW) Ϫ1 h Ϫ1 at growth rates of between 0.40 and 0.36 h Ϫ1 (71), whereas styrene epoxidation caused pronounced acetate formation already at glucose uptake rates of above 2 mmol (g CDW) Ϫ1 h Ϫ1 and a low growth rate of 0.1 h Ϫ1 .…”