Identification of mannitol as compatible solute in Gluconobacter oxydans

Abstract

Gluconobacter oxydans is an industrially important bacterium owing to its regio- and enantio-selective incomplete oxidation of various sugars, alcohols, and polyols. The complete genome sequence is available, but it is still unknown how the organism adapts to highly osmotic sugar-rich environments. Therefore, the mechanisms of osmoprotection in G. oxydans were investigated. The accumulation and transport of solutes are hallmarks of osmoadaptation. To identify potential osmoprotectants, G. oxydans was grown on a yeast glucose medium in the presence of 100 mM potassium phosphate (pH 7.0) along with various concentrations of sucrose (0-600 mM final concentration), which was not metabolized. Intracellular metabolites were analyzed by HPLC and (13)C NMR spectroscopy under stress conditions. Both of these analytical techniques highlighted the accumulation of mannitol as a potent osmoprotectant inside the stressed cells. This intracellular mannitol accumulation correlated with increased extracellular osmolarity of the medium. For further confirmation, the growth behavior of G. oxydans was analyzed in the presence of small amounts of mannitol (2.5-10 mM) and 300 mM sucrose. Growth under sucrose-induced osmotic stress conditions was almost identical to control growth when exogenous mannitol was added in low amounts. Thus, mannitol alleviates the osmotic stress of sucrose on cellular growth. Moreover, the positive effect of exogenous mannitol on the rate of glucose consumption and gluconate formation was also monitored. These results may be helpful to optimize the processes of industrial product formation in highly concentrated sugar solutions.

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

“…These values were in the same range as for the wild type without osmotic stress (t d = 2 h, final OD 600 = 2.0). Hence, low concentrations of mannitol could alleviate osmotic stress from the cells imposed by sucrose (Zahid et al 2015). The results indicate that the growth retardation observed for the Δgox1432 mutant and the double mutant under osmotic stress was due to defects in mannitol synthesis.…”

“…Recently, we have reported the role of D-mannitol as an osmoprotectant in G. oxydans 621H (Zahid et al 2015). Mannitol functions as an osmolyte in plants and fungi (Jennings 1984;Grant 2004;Empadinhas and da Costa 2009) as well as in different bacteria like Pseudomonas fluorescence (Kets et al 1996) and A. baylyi (Sand et al 2013) and in hetero-fermentative lactic acid bacteria (Wisselink et al 2002).…”

“…The wild-type and the mannitol dehydrogenase deletion mutants were cultivated in YGP medium containing 450 mM sucrose. Analysis of intracellular mannitol content was performed as described by Zahid et al (2015) wild type. NADH-dependent reduction of fructose in G. oxydans Δgox0849 was 2-fold lower compared to the wild type.…”

“…Glucose and gluconate concentrations were quantified according to standard calibration curves. Analysis of intracellular mannitol content was performed as described by Zahid et al (2015).…”

“…Mannitol is the most abundant polyol in nature and is used as an osmoprotectant mostly in eukaryotes such as plants and fungi (Empadinhas and da Costa 2008). In a previous study, it was shown that the industrially important microorganism, Gluconobacter oxydans, copes with osmotic stress by synthesizing mannitol inside the cell (Zahid et al 2015). The organism accumulates mannitol as a compatible solute in media with high concentrations of sucrose, glucose, or fructose (Table S1).…”