Comparative studies ofEscherichia coli strains using different glucose uptake systems: Metabolism and energetics

Chen, Ruizhen; Yap, Wyanda M. G. J.; Postma, Pieter W.; Bailey, James E.

doi:10.1002/(sici)1097-0290(19971205)56:5<583::aid-bit12>3.0.co;2-d

Cited by 41 publications

(9 citation statements)

References 13 publications

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“…The Malt enzyme originates from the Mal32 gene encoding an α-glucosidase in Saccharomyces cerevisiae ; the gene sequence was codon-optimized for bacterial expression and gives rise to a ∼71 kDa protein of 615 residues with an estimated net charge of −2.4 at pH 7.5. , Maltase (alpha-glucosidase; EC 3.2.1.20) hydrolyzes the central α-1,4 glycosidic bond in maltose, yielding two glucose units. The gene encoding Glk (343 residues, ∼37 kDa, net charge of −2.1 at pH 7.5) was cloned directly from Escherichia coli strain BL21(DE3) and expressed as described elsewhere. , Glucokinase or hexokinase (EC 2.7.1.2) phosphorylates glucose to glucose-6-phosphate as the entry step in glycolysis . Am and Malt are both monomeric proteins, while Glk is predicted to be a dimer.…”

Section: Resultsmentioning

confidence: 99%

“…The overall reaction scheme is summarized in Scheme ,where S 7 is maltoheptaose, S 3 is maltotriose, S 2 is maltose, S 1 is glucose, Gp is glucose-6-phosphate, Pgl is 6-phospho- d -glucono-1,5-lactone, NAD/NADH are the oxidized and reduced forms of nicotinamide adenine dinucleotide (respectively), ATP is adenosine triphosphate, and ADP is adenosine diphosphate. Although Glk technically has two reactants, ATP was always added at a vast excess to maintain saturating conditions justifying use of the Michaelis–Menten model for overall analysis. , G6PDH was included as a coupled enzymatic reporter, always in solution and in large excess. A maltoheptaose substrate was used to monitor turnover by the three-enzyme cascade directly or to characterize the individual enzymes simply by including all the other required enzymes in excess in solution.…”

Section: Resultsmentioning

confidence: 99%

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Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

et al. 2019

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Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway’s kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

et al. 2019

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“…In addition, accumulation of other compounds such as lactate, formate, pyruvate, ethanol etc. has been observed [7,13,17]. Although excretion of many other compounds besides 'well-known' ones e.g .…”

Section: Introductionmentioning

confidence: 99%

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

2011

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BackgroundGrowth 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 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.ResultsWe 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 μ and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing μ 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 μ, 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.ConclusionsIt 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.

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“…As detailed below each individual enzyme will be treated as following a Michaelis–Menten (MM) model, adding excess cofactors (e.g. ATP and NAD + ), so as to make them treatable with the MM formula for their specific substrate. , To characterize the flux of the entire cascade we utilized MM fittings of the final product formation, utilizing the maximum/initial velocity of each experiment, which was tracked by observing increasing 340 nm absorbance due to NADH formation, as a function of the initial substrate (maltoheptaose) concentration. The analysis is in line with previous work with this enzyme cascade; , details are available in the Materials and Methods section and a schematic is shown in Figure B.…”

Section: Resultsmentioning

confidence: 99%

Gold Nanoparticle Templating Increases the Catalytic Rate of an Amylase, Maltase, and Glucokinase Multienzyme Cascade through Substrate Channeling Independent of Surface Curvature

Dı́az

Choo

et al. 2020

ACS Catal.

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The templating of enzymes has shown myriad advantages, including increased stability and kinetic rates. Specifically, the use of nanoparticles (NPs) as templates has been shown to increase the kinetic rates over larger macroscale scaffolds, in part by overcoming diffusion limitations. Within the field, there is considerable debate over the parameters that optimize this interaction, e.g., NP size, curvature, and surface ligands. Recently much interest has been seen in the pursuit of complete enzyme cascades on nanoscaffolds, with the increased enzyme proximity providing multiple benefits. In this work we demonstrate that multienzyme cascades can be templated on individual gold NPs. Utilizing a three-enzyme cascade of amylase, maltase, and glucokinase, we found a ∼3-fold enhancement in product formation when all three enzymes were bound to the same NP as compared to when the enzymes were bound separately and then combined as well as freely diffusing in solution. This strongly suggests that the increased kinetics was due to substrate channeling. In addition, we investigated the effect of size and curvature on the kinetics of the cascade by using different-shaped gold NPs. Substrate channeling was observed in small and large gold nanospheres (∼30 and 70 nm diameters, respectively) and gold nanostars with an equivalent surface area. Equivalent enhanced kinetics for the enzyme cascade were observed for all of the tested nanoscaffolds, in contrast with what has been reported for single enzymes on gold NPs where changes in curvature resulted in modified kinetics. The results of this work should allow for optimized design of NPs and enzyme cascades for in vitro biocatalysis.

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Comparative studies ofEscherichia coli strains using different glucose uptake systems: Metabolism and energetics

Cited by 41 publications

References 13 publications

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

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

Gold Nanoparticle Templating Increases the Catalytic Rate of an Amylase, Maltase, and Glucokinase Multienzyme Cascade through Substrate Channeling Independent of Surface Curvature

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