Metabolic and transcriptional response of Escherichia coli with a NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans
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The NAD+-dependent glyceraldehyde-3-phosphate-dehydrogenase (NAD+-GAPDH) is a key enzyme to sustain the glycolytic function in Escherichia coli and to generate NADH. In the absence of NAD+-GAPDH activity, the glycolytic function can be restored through NADP+-dependent GAPDH heterologous expression. Here, some metabolic and transcriptional effects are described when the NAD+-GAPDH gene from E. coli (gapA) is replaced with the NADP+-GAPDH gene from Streptococcus mutans (gapN). Expression of gapN was controlled by the native gapA promoter (E. coliΔgapA::gapN) or by the constitutive trc promoter in a multicopy plasmid (E. coliΔgapA::gapN/pTrcgapN). The specific NADP+-GAPDH activity was 4.7 times higher in E. coliΔgapA::gapN/pTrcgapN than E. coliΔgapA::gapN. Growth, glucose consumption and acetic acid production rates increased in agreement with the NADP+-GAPDH activity level. Analysis of E. coliΔgapA::gapN/pTrcgapN showed that although gapN expression complemented NAD+-GAPDH activity, the resulting low NADH levels decreased the expression of the respiratory chain and oxidative phosphorylation genes (ndh, cydA, cyoB and atpA). In comparison with the wild type strain, E. coliΔgapA::gapN/pTrcgapN decreased the percentage of mole of oxygen consumed per mole of glucose metabolized by 40 % with a concomitant reduction of 54 % in the ATP/ADP ratio. The cellular response to avoid NADPH excess led to the overexpression of the transhydrogenase coded by udhA and the down-regulation of the pentose-phosphate and Krebs cycle genes, which reduced the CO2 production and increased the acetic acid synthesis. The E. coli strains obtained in this work can be useful for future metabolic engineering efforts aiming for the production of metabolites which biosynthesis depends on NADPH.
KeywordsEscherichia coli NAD(P)+-dependent glyceraldehyde-3-phosphate-dehydrogenase Respiration ATP NAD(P)(H)
We gratefully acknowledge Georgina Hernández-Chávez, Luz María Martínez, Mercedes Enzaldo, Ramón de Anda and Adelfo Escalante for technical assistance. We thank Martha Giles (Facultad de Química—Universidad Nacional Autónoma de México) for providing the Streptococcus mutans strain. This work was supported by the Mexican Council of Science and Technology (CONACyT). SC-L held a scholarship from CONACyT.
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