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Here we report the first metatranscriptomic analysis of gene expression and regulation of ‘ Candidatus Accumulibacter’-enriched lab-scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl-CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen. [ABSTRACT FROM AUTHOR]

Background: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism., Results: Overexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I ( pfkA ) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this Δ pfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the Δ pfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The Δ pfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the Δ pfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13 C-pulse experiments in the Δ pfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium)., Conclusions: We engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.

Metabolic flux analysis via (13)C labeling ((13)C MFA) quantitatively tracks metabolic pathway activity and determines overall enzymatic function in cells. Three core techniques are necessary for (13)C MFA: (1) a steady state cell culture in a defined medium with labeled-carbon substrates; (2) precise measurements of the labeling pattern of targeted metabolites; and (3) evaluation of the data sets obtained from mass spectrometry measurements with a computer model to calculate the metabolic fluxes. In this review, we summarize recent advances in the (13)C-flux analysis technologies, including mini-bioreactor usage for tracer experiments, isotopomer analysis of metabolites via high resolution mass spectrometry (such as GC-MS, LC-MS, or FT-ICR), high performance and large-scale isotopomer modeling programs for flux analysis, and the integration of fluxomics with other functional genomics studies. It will be shown that there is a significant value for (13)C-based metabolic flux analysis in many biological research fields., ((c) 2009 Wiley Periodicals, Inc.)