title: Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli.
abstract: In Escherichia 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 an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Δpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf 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 in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, 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.
authors.affiliation: Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: K
authors.initials: K
authors.lastname: Olavarria
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: J
authors.initials: J
authors.lastname: De Ingeniis
authors.affiliation: Department of Bioengineering, University of California San Diego, San Diego, CA, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Zielinski
authors.affiliation: Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: M
authors.initials: M
authors.lastname: Fuentealba
authors.affiliation: Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Muñoz
authors.affiliation: Department of Bioengineering, University of California San Diego, San Diego, CA, USA.
authors.forename: D
authors.initials: D
authors.lastname: McCloskey
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.|Department of Bioengineering, University of California San Diego, San Diego, CA, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Feist
authors.affiliation: Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Cabrera
mesh_terms: D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009243:NAD / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D049490:Systems Biology
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D005954:Glucosephosphate Dehydrogenase