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.

Salt induction and activation of MtlD, the key enzyme in the synthesis of the compatible solute mannitol in Acinetobacter baumannii

“…It seems that A. baumannii cannot easily compensate for the loss of mannitol by increasing the production of another solute, as for example, glutamate. This is consistent with the finding in G. oxydans , where a mutant defective in mannitol production was inhibited in growth at high osmolarities (Zahid & Deppenmeier, ). G. oxydans can, in addition, take up mannitol from the medium and exogenous mannitol restores growth of the mannitol biosynthesis mutant.…”

Global Analysis of Mannitol 2-Dehydrogenase in Lactobacillus reuteri CRL 1101 during Mannitol Production through Enzymatic, Genetic and Proteomic Approaches

“…When this bacterium was cultured in the presence of mannitol as the sole carbon source, five genes coding for the proteins MDH and fructokinase, and an ATP binding cassette (ABC) transporter complex were induced suggesting the organization into one operon. In another study, the expression of both enzymes NADH- and NADPH-dependent MDH has been evaluated in the bacterium Gluconobacter oxydans growing under osmotic stress conditions in the presence of sucrose [38]. NADPH-dependent MDH was determinant for mannitol production as the mRNA abundance of this enzyme was 30-fold higher than the NADH-dependent enzyme.…”

Degradation of the low-calorie sugar substitute 5-ketofructose by different bacteria

“…6 were heterologously produced in E. coli and purified by Strep-tag affinity chromatography. It became evident that the 5-ketogluconate reducing enzyme GOX1432 (Zahid and Deppenmeier 2016 ) and the α-ketocarbonyl reductase GOX0644 (Schweiger et al 2010 ) were able to reduce 5-KF with NADPH as reductant (Fig. 6 ).…”