title: Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.
abstract: Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP+-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD+as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP+-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP+-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.
authors.affiliation: Institute of Microbiology and Biotechnology, Meckenheimer Allee 168, 53115, Bonn, Germany.
authors.forename: Nageena
authors.initials: N
authors.lastname: Zahid
authors.affiliation: Institute of Microbiology and Biotechnology, Meckenheimer Allee 168, 53115, Bonn, Germany. udeppen@uni-bonn.de.
authors.forename: Uwe
authors.identifier: http://orcid.org/0000-0002-7810-7663
authors.initials: U
authors.lastname: Deppenmeier
mesh_terms: D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D020869:Gene Expression Profiling; D005816:Genetic Complementation Test; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008353:Mannitol / Q000378:metabolism*; D008354:Mannitol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D064587:Osmoregulation; D009997:Osmotic Pressure; D013312:Stress, Physiological
chemical_list: D003470:Culture Media; D008353:Mannitol; D008354:Mannitol Dehydrogenases
keywords: Biotransformation; Compatible solute; Fructose reduction; Osmotic stress; Vitamin C