RRC ID 3232
Author Zhu J, Shimizu K.
Title The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli.
Journal Appl. Microbiol. Biotechnol.
Abstract The effect of gene knockout on metabolism in the pflA-, pflB-, pflC-, and pflD- mutants of Escherichia coli was investigated. Batch cultivations of the pfl- mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA- and pflB- mutants, but not pflC- and pflD- mutants, produced large amounts of D-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA- and pflB- mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl- mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.
Volume 64(3)
Pages 367-75
Published 2004-4
DOI 10.1007/s00253-003-1499-9
PMID 14673546
MeSH Acetate Kinase / metabolism Acetyltransferases / genetics* Adenosine Triphosphate / metabolism Aerobiosis Alcohol Dehydrogenase / metabolism Energy Metabolism Escherichia coli / enzymology* Escherichia coli / genetics* Escherichia coli / metabolism Fructose / metabolism Genes, Bacterial Gluconates / metabolism Glucose / metabolism Glucosephosphate Dehydrogenase / metabolism Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating) / metabolism Glycerol / metabolism Glycolysis Isomerism L-Lactate Dehydrogenase / metabolism Lactic Acid / analysis* Lactic Acid / biosynthesis* Lactic Acid / chemistry Mutation Phosphoenolpyruvate Carboxylase / metabolism Phosphogluconate Dehydrogenase / metabolism Pyruvate Kinase / metabolism Pyruvic Acid / metabolism
IF 3.67
Times Cited 80
Prokaryotes E. coli JW0885 JW0886 JW3923 JW3924