Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene

FEBS Letters

Losada, Manuel¹  Serrano, Aurelio¹  Valverde, Federico¹ 

[1] Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla-CSIC, Avda. Américo Vespucio s/n, E-41092 Sevilla, Spain

关键词: Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase;    Functional complementation;    Glycolysis;    Gluconeogenesis;    Metabolic engineering;    GAPDH;    D-glyceraldehyde-3-phosphate dehydrogenase;    G3P;    D-glyceraldehyde-3-phosphate;    3-PGA;    D-3-phosphoglycerate;    DHAP;    dihydroxyacetone-phosphate;    1;    3-BPGA;    D-1;    3-bisphosphoglycerate;    IPTG;    isopropyl-β-D-thiogalactopyranoside;    NPA;    p-nitrophenyl acetate;   

DOI  :  10.1016/S0014-5793(99)00430-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown