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Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers

Paper ID Volume ID Publish Year Pages File Format Full-Text
31999 44872 2007 16 PDF Available
Title
Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers
Abstract

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. succinogenes [1-13C]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. succinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. succinogenes succinate production are discussed.

Keywords
Actinobacillus succinogenes; Succinate; Metabolic flux analysis; Biomass composition; Oxaloacetate decarboxylase; Malic enzyme; Transhydrogenase
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Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 9, Issue 2, March 2007, Pages 177–192
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us