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

• Published in: Journal of biotechnology Vol. 122; no. 2; pp. 254 - 266
• Main Authors: Li, Mai, Ho, Pei Yee, Yao, Shanjing, Shimizu, Kazuyuki
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 23-03-2006; Amsterdam Elsevier; New York, NY
• Subjects: 13C-labeling experiment; Biological and medical sciences; Biological Transport; Biotechnology; Carbohydrate Dehydrogenases - genetics; Carbon Isotopes - analysis; Dihydrolipoamide Dehydrogenase - genetics; Enzyme activity; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Fundamental and applied biological sciences. Psychology; Ketoglutarate Dehydrogenase Complex - genetics; lpdA gene knockout; Metabolic flux analysis; Metabolic regulation; Mutation; Pyruvate Dehydrogenase Complex - genetics; Pyruvate Dehydrogenase Complex - metabolism; Metabolic regulation; Enzyme activity; 13C-labeling experiment; lpdA gene knockout; Metabolic flux analysis; Metabolite; Escherichia coli; Concentration; Metabolism; Regulation(control); Enzymatic activity; Gene; Tagging; Bacteria; Intracellular; Mutation; Enterobacteriaceae
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2005.09.016

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.