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Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments

Paper ID Volume ID Publish Year Pages File Format Full-Text
25806 43602 2006 13 PDF Available
Title
Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments
Abstract

The lipoamide dehydrogenase (LPD) encoded by lpdA gene is a component of the pyruvate dehydrogenase complex (PDHc), α-ketoglutarate dehydrogenase (AKGDH) and the glycine cleavage multi-enzyme (GCV) systems. In the present study, cell growth characteristics, enzyme activities and intracellular metabolite concentrations were compared between the parent strain Escherichia coli BW25113 and its lpdA knockout mutant in batch and continuous cultures. The lpdA knockout mutant produced significantly more pyruvate and l-glutamate under aerobiosis. Some d-lactate and succinate also accumulated in the culture broth. Based on the investigation of enzyme activities and intracellular metabolite concentrations, acetyl-CoA was considered to be formed by the combined reactions through pyruvate oxidase (PoxB), acetyl-CoA synthetase (Acs) and acetate kinase (Ack)-phosphoacetyltransferase (Pta) in the lpdA mutant. The effect of the lpdA gene knockout on the intracellular metabolic flux distributions was investigated based on 1H–13C NMR spectra and GC–MS signals obtained from 13C-labeling experiment using the mixture of [U-13C] glucose, [1-13C] glucose, and naturally labeled glucose. Flux analysis of the lpdA mutant indicated that the Entner-Doudoroff (ED) pathway and the glyoxylate shunt were activated. The fluxes through glycolysis and oxidative pentose phosphate (PP) pathway (except for the flux through glucose-6-phosphate dehydrogenase) were slightly downregulated. The TCA cycle was also downregulated in the mutant strain. On the other hand, the fluxes through the anaplerotic reactions of PEP carboxylase, PEP carboxykinase and malic enzyme were upregulated, which were consistent with the results of enzyme activities. Furthermore, the influence of the poxB gene knockout on the growth of E. coli was also studied because of its similar function to PDHc which connects the glycolysis to the TCA cycle. Under aerobiosis, a comparison of lpdA mutant and poxB mutant indicated that PDHc is the main enzyme which catalyzes the reaction from pyruvate to acetyl-CoA in the parent strain, while PoxB plays a very important role in the PDHc-deficient strain.

Keywords
lpdA gene knockout; Enzyme activity; 13C-labeling experiment; Metabolic flux analysis; Metabolic regulation
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Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 122, Issue 2, 23 March 2006, Pages 254–266
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