13C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in Escherichia coli

Phenol is an important chemical product that can be used in a wide variety of applications, and it is currently produced from fossil resources. Fermentation production of phenol from renewable biomass resources by microorganisms is highly desirable for sustainable development. However, phenol toxicity hampers phenol production in industrial microorganisms such as Escherichia coli. In the present study, it was revealed that culturing E. coli in the presence of phenol not only decreased growth rate, but also biomass yield. This suggests that phenol affects the carbon flow of the metabolism, but the mechanism is unknown. To investigate the effect of phenol on the flux distribution of central carbon metabolism, 13C-metabolic flux analysis (13C-MFA) was performed on cells grown under different phenol concentrations (0, 0.1, and 0.15%). 13C-MFA revealed that the TCA cycle flux reduced by 25% increased acetate production from acetyl-CoA by 30% in the presence of 0.1% phenol. This trend of flux changes was emphasized at a phenol concentration of 0.15%. Although the expression level of citrate synthase, which catalyzes the first reaction of the TCA cycle, does not change regardless of phenol concentrations, the in vitro enzyme activity assay shows that the reaction was inhibited by phenol. These results suggest that the TCA cycle flux decreased due to phenol inhibition of citrate synthase; therefore, ATP could not be sufficiently produced by respiration, and growth rate decreased. Furthermore, since carbon was lost as acetate due to overflow metabolism, the biomass yield became low in the presence of phenol

Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks

Vesicle-Like Biomechanics Governs Important Aspects of Nuclear Geometry in Fission Yeast

It has long been known that during the closed mitosis of many unicellular eukaryotes, including the fission yeast (Schizosaccharomyces pombe), the nuclear envelope remains intact while the nucleus undergoes a remarkable sequence of shape transformations driven by elongation of an intranuclear mitotic spindle whose ends are capped by spindle pole bodies embedded in the nuclear envelope. However, the mechanical basis of these normal cell cycle transformations, and abnormal nuclear shapes caused by intranuclear elongation of microtubules lacking spindle pole bodies, remain unknown. Although there are models describing the shapes of lipid vesicles deformed by elongation of microtubule bundles, there are no models describing normal or abnormal shape changes in the nucleus. We describe here a novel biophysical model of interphase nuclear geometry in fission yeast that accounts for critical aspects of the mechanics of the fission yeast nucleus, including the biophysical properties of lipid bilayers, forces exerted on the nuclear envelope by elongating microtubules, and access to a lipid reservoir, essential for the large increase in nuclear surface area during the cell cycle. We present experimental confirmation of the novel and non-trivial geometries predicted by our model, which has no free parameters. We also use the model to provide insight into the mechanical basis of previously described defects in nuclear division, including abnormal nuclear shapes and loss of nuclear envelope integrity. The model predicts that (i) despite differences in structure and composition, fission yeast nuclei and vesicles with fluid lipid bilayers have common mechanical properties; (ii) the S. pombe nucleus is not lined with any structure with shear resistance, comparable to the nuclear lamina of higher eukaryotes. We validate the model and its predictions by analyzing wild type cells in which ned1 gene overexpression causes elongation of an intranuclear microtubule bundle that deforms the nucleus of interphase cells

Identification of alcohol stress tolerance genes of Synechocystis sp. PCC 6803 using adaptive laboratory evolution

Abstract Background Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance. Results Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain. Conclusions Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803

Investigation of useful carbon tracers for 13C-metabolic flux analysis of Escherichia coli by considering five experimentally determined flux distributions

The 13C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13C-labeled glucose for 13C-metabolic flux analysis (13C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli, the best fitted flux distribution and the 95% confidence interval were estimated by the 13C-MFA procedure. A comparison of the precision scores showed that [1, 2-13C]glucose and a mixture of [1-13C] and [U-13C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1-13C], and [U-13C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13C-MFA experiments. Keywords: 13C-metabolic flux analysis, Design of experiment, 13C-labeling experiment, Escherichia coli, Computer simulatio

MOESM1 of Identification of alcohol stress tolerance genes of Synechocystis sp. PCC 6803 using adaptive laboratory evolution

Additional file 1. Additional figures, table, and details procedures for strain construction