Format

Send to

Choose Destination
Microbiology. 2014 Dec;160(Pt 12):2780-93. doi: 10.1099/mic.0.082180-0. Epub 2014 Sep 22.

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli.

Author information

1
Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
2
Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
3
Department of Bioengineering, University of California San Diego, San Diego, CA, USA.
4
Department of Bioengineering, University of California San Diego, San Diego, CA, USA Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark ricabrer@uchile.cl afeist@ucsd.edu.
5
Facultad de Ciencias, Universidad de Chile, Santiago, Chile ricabrer@uchile.cl afeist@ucsd.edu.

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.

PMID:
25246670
DOI:
10.1099/mic.0.082180-0
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Ingenta plc
Loading ...
Support Center