Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene.
A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3-phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde-3-phosphate dehydrogenases. According to the irreversible catabolic character of the non-phosphorylating reaction, the GapN-complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden-Meyerhof pathway with no net energy yield is feasible.
Valverde
F
F
Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla-CSIC, Spain.
Losada
M
M
Serrano
A
A
D004926:Escherichia coli / Q000235:genetics*; D017343:Genes, Plant; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D006019:Glycolysis; D016296:Mutagenesis; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases