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Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting.

ABSTRACT: BACKGROUND: Growth substrates, aerobic/anaerobic conditions, specific growth rate (?) etc. strongly influence Escherichia coli cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA. RESULTS: We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor E. coli K-12 MG1655 flux and energy metabolism dynamic responses to change of ? and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing ? and carbon wasting to non-CO2 by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of E. coli pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on ?, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway. CONCLUSIONS: It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.

SUBMITTER: Valgepea K

PROVIDER: S-EPMC3149000 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting.

Valgepea Kaspar K Adamberg Kaarel K Vilu Raivo R

BMC systems biology 20110705

<h4>Background</h4>Growth substrates, aerobic/anaerobic conditions, specific growth rate (μ) etc. strongly influence Escherichia coli cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance ca ...[more]

PMID: 21726468

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Project description:Biosynthesis of poly-3-hydroxybutyrate (PHB) as a fermentation product enables the coupling of growth and product generation. Moreover, the reduction of oxygen supply should reduce operative cost and increase product yield. Generation of PHB as a fermentation product depends on the in vivo activity of an NADH-preferring acetoacetyl-CoA reductase. Proof of this concept requires (i) quantification of the cofactor preference, in physiologically relevant conditions, of a putative NADH-preferring acetoacetyl-CoA reductase and (ii) verification of PHB accumulation using an NADH-preferring acetoacetyl-CoA reductase in a species naturally incapable of doing so, for example, Escherichia coli. This dataset contains kinetic data obtained by spectrophotometry and data from a continuous culture of an engineered E. coli strain accumulating PHB under oxygen-limiting conditions. In this dataset it is possible to find (1) enzyme stability assays; (2) initial rates and progress curves from reactions catalyzed by two acetoacetyl-CoA reductases; (3) estimations of the relative use of NADH and NADPH by two acetoacetyl-CoA reductases; (4) estimations of the flux capacity of the reaction catalyzed by an acetoacetyl-CoA reductase; (5) biomass composition of an engineered E. coli strain transformed with a plasmid; (6) calculation of reconciled specific rates of this engineered strain growing on sucrose as the sole carbon source under oxygen limitation and (7) metabolic fluxes distributions during the continuous growth of this engineered strain. Because a relatively small number of acetoacetyl-CoA reductases have been kinetically characterized, data and scripts here provided could be useful for further kinetic characterizations. Moreover, the procedure described to estimate biomass composition could be interesting to estimate plasmid and protein burden in other strains. Application of data reconciliation to fermentations should help to obtain specific rates consistent with the principle of mass and electron conservation. All the required data and scripts to perform these analyses are deposited in a Mendeley Data repository. This article was co-submitted with the manuscript entitled "An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichiasia. coli".

Dataset Information

Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting.

Publications

Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting.

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