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

Maeda, Kousuke; Okahashi, Nobuyuki; Toya, Yoshihiro; Mizutani, Fumio; Shimizu, Hiroshi

doi:10.1016/j.meteno.2016.06.001

Cited by 16 publications

(15 citation statements)

References 39 publications

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“…Multiple good resources are available for metabolic flux analysis (MFA) with 13 C-glucose as the input 16 , 17 . Although many studies have looked at central carbon metabolism using labeled glucose, different molecules can be biosynthesized from these pathways via a variety of transformations.…”

Section: Discussionmentioning

confidence: 99%

A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways

Walvekar¹,

Rashida²,

Maddali³

et al. 2018

Wellcome Open Res

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Liquid chromatography-mass spectrometry (LC-MS/MS) based approaches are widely used for the identification and quantitation of specific metabolites, and are a preferred approach towards analyzing cellular metabolism. Most methods developed come with specific requirements such as unique columns, ion-pairing reagents and pH conditions, and typically allow measurements in a specific pathway alone. Here, we present a single column-based set of methods for simultaneous coverage of multiple pathways, primarily focusing on central carbon, amino acid, and nucleotide metabolism. We further demonstrate the use of this method for quantitative, stable isotope-based metabolic flux experiments, expanding its use beyond steady-state level measurements of metabolites. The expected kinetics of label accumulation pertinent to the pathway under study are presented with some examples. The methods discussed here are broadly applicable, minimize the need for multiple chromatographic resolution methods, and highlight how simple labeling experiments can be valuable in facilitating a comprehensive understanding of the metabolic state of cells.

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Section: Discussionmentioning

confidence: 99%

A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways

Walvekar¹,

Rashida²,

Maddali³

et al. 2018

Wellcome Open Res

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“…A computer simulation was conducted for the design of the 13 C-MFA experiment as previously described [ 9 ]. The intracellular flux distribution data was determined using the modified literature data through metabolic flux balancing [ 6 ].…”

Section: Methodsmentioning

confidence: 99%

“…The computational procedure for 13 C-MFA was performed using a Python version of OpenMebius implemented in Python 2.7.9 [ 9 , 21 ], by which 22 independent fluxes were iteratively tuned by minimizing the residual between the experimental and simulated 13 C-enrichments of proteinogenic amino acid fragments. Furthermore, the 13 C-labeling patterns of CO 2 were independently optimized in the flux estimation.…”

Section: C-metabolic Flux Analysismentioning

confidence: 99%

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-l-methionine production

2018

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BackgroundSaccharomyces cerevisiae is a host for the industrial production of S-adenosyl-l-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, 13C-metabolic flux analysis (13C-MFA) was conducted to investigate the metabolic regulation responsible for the high SAM production from ethanol.ResultsThe comparison between the metabolic flux distributions of central carbon metabolism showed that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that of glucose. Estimates of the ATP balance from the 13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose.ConclusionsThese results suggest that the enhanced ATP regeneration due to ethanol assimilation was critical for the high SAM accumulation.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0935-6) contains supplementary material, which is available to authorized users.

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“…The regulatory patterns of acetate consumption and glyoxylate cycle remain to be described for Salmonella, as well as the role of the isocitrate dehydrogenase in this organism. According to Maeda et al (2016), the mixture of non-labeled, [1-13 C] and [U-13 C] glucose at ratios of 4:1:5 could be applied to estimate the metabolic fluxes through glyoxylate pathway with higher precision.…”

Section: Impact Of Growth Rate On Central Carbon Metabolic Flux Distrmentioning

confidence: 99%

Mapping Salmonella typhimurium pathways using 13C metabolic flux analysis

Correia

Sargo

Silva

et al. 2019

Metabolic Engineering

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In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13 C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13 C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genomescale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.

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

Cited by 16 publications

References 39 publications

A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways

A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-l-methionine production

Mapping Salmonella typhimurium pathways using 13C metabolic flux analysis

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