1887

Abstract

In , 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 (Δ). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring G6PDH. Through homologous recombination, the loci (encoding G6PDH) in the chromosomes of WT and Δ strains were replaced by DNA encoding G6PDH. Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Δ strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the locus showed that the expression level of the mutant enzyme was similar to the native enzyme 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 modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, , the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden–Meyerhof pathway. Interestingly, a genomic database search suggested 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.

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2014-12-01
2020-07-13
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