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...

The ability of biological systems to adapt to genetic and environmental perturbations is a fundamental but poorly understood process at the molecular level. By quantifying metabolic fluxes and global mRNA abundance, we investigated the genetic and metabolic mechanisms that underlie adaptive evolution of four metabolic gene deletion mutants of Escherichia coli (⌬pgi, ⌬ppc, ⌬pta, and ⌬tpi) in parallel evolution experiments of each mutant. The initial response to the gene deletions was flux rerouting through local bypass reactions or normally latent pathways. The principal effect of evolution was improved capacity of already active pathways, whereas new flux distributions were not observed. Combinatorial changes in capacity and pathway activation, however, led to different intracellular flux states that enabled evolution in three of the four parallel cases tested. The molecular bases of the evolved phenotypes were then elucidated by global mRNA transcript analyses. Activation of latent pathways and flux changes in the tricarboxylic acid cycle were found to correlate well with molecular changes at the transcriptional level. Flux alterations in other central metabolic pathways, in contrast, were apparently not connected to changes in the transcriptional network. These results give new insight into the dynamics of the evolutionary process by demonstrating the flexibility of the metabolic network of E. coli to compensate for genetic perturbations and the utility of combining multiple high throughput data sets to differentiate between causal and noncausal mechanistic changes.

Network topology is a necessary fundament to understand function and properties of microbial reaction networks. A valuable method for experimental elucidation of metabolic network topology is metabolic flux ratio analysis, which quantifies the relative contribution of two or more converging pathways to a given metabolite. It is based on 13C-labeling experiments, gas chromatography-mass spectrometry analysis, and probabilistic equations that relate mass distributions in proteinogenic amino acids to pathway activity. Here, we describe the protocol for sample generation and illustrate the principles underlying the calculation of metabolic flux ratios with three examples. These principles are also implemented in the publicly available software FiatFlux, which directly calculates flux ratios from the mass spectra of amino acids.

The authors analyze the role transcription plays in regulating bacterial metabolic flux. Of 91 transcriptional regulators studied, 2/3 affect absolute fluxes, but only a small number of regulators control the partitioning of flux between different metabolic pathways.

Azides are building blocks of increasing importance in synthetic chemistry, chemical biology, and materials science. Azidobenziodoxolone (ABX, Zhdankin reagent) is a valuable azide source, but its safety profile has not been thoroughly established. Herein, we report a safety study of ABX, which shows its hazardous nature. We introduce two derivatives, tBu-ABX and ABZ (azidobenziodazolone), with a better safety profile, and use them in established photoredox- and metal-mediated azidations, and in a new ring-expansion of silylated cyclobutanols to give azidated cyclopentanones.