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 n on-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli str ain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydroge nase, to grow aerobically on sugars. The functionally complemented mutant e xhibited high levels of the catalytically active plant enzyme, which render s 3-phosphoglycerate and NADPH, thus bypassing the first substrate level ph osphorylation step of the glycolysis. As expected if such a glycolytic bypa ss would be operative in vivo, this clone failed to grow anaerobically on s ugars in contrast to W3CG clones complemented with phosphorylating glyceral dehyde-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. (C) 1999 Federation of European Biochemical Societies.