|Title||The Metabolic Impact of a NADH-producing Glucose-6-phosphate Dehydrogenase in Escherichia coli.|
|Publication Type||Journal Article|
|Year of Publication||2014|
|Authors||Olavarria K, De Ingeniis J, Zielinski DC, Fuentealba M, Muñoz R, McCloskey D, Feist AM, Cabrera R|
In Escherichia coli (E. coli), the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by a NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDHR46E,Q47E. Through homologous recombination, the zwf loci (encoding for G6PDH) in the chromosomes of wild type (wt) and Δpgi E. coli strains were replaced by DNA encoding for LmG6PDHR46E,Q47E. Contrary to the predictions of a Robustness Analysis performed by flux balances simulation, the replacements caused a substantial effect on the growth rates, increasing 59% in the Δpgi strain, while falling 44% in the wt. The quantitative PCR (qPCR) for the locus zwf showed that the expression level of the mutant enzyme is similar to the native, and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modeling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by the G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggests that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.
|Alternate Journal||Microbiology (Reading, Engl.)|
The Metabolic Impact of a NADH-producing Glucose-6-phosphate Dehydrogenase in Escherichia coli.