Efficient mannitol production by wild-type Lactobacillus reuteri CRL 1101 is attained at constant pH using a simplified culture medium.

Mannitol is a natural polyol with multiple industrial applications. In this work, mannitol production by Lactobacillus reuteri CRL 1101 was studied at free- and controlled-pH (6.0-4.8) fermentations using a simplified culture medium containing yeast and beef extracts and sugarcane molasses. The activity of mannitol 2-dehydrogenase (MDH), the enzyme responsible for mannitol synthesis, was determined. The effect of the initial biomass concentration was further studied. Mannitol production (41.5 ± 1.1 g/l), volumetric productivity (Q Mtl 1.73 ± 0.05 g/l h), and yield (Y Mtl 105 ± 11 %) were maximum at pH 5.0 after 24 h while the highest MDH activity (1.66 ± 0.09 U/mg protein) was obtained at pH 6.0. No correlation between mannitol production and MDH activity was observed when varying the culture pH. The increase (up to 2000-fold) in the initial biomass concentration did not improve mannitol formation after 24 h although a 2-fold higher amount was produced at 8 h using 1 or 2 g cell dry weight/l comparing to the control (0.001 g cell dry weight/l). Finally, mannitol isolation under optimum fermentation conditions was achieved. The mannitol production obtained in this study is the highest reported so far by a wild-type L. reuteri strain and, more interestingly, using a simplified culture medium.

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis.

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress.

Spontaneous formation of a mannitol-producing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose.

We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown.

Immunolocalization of mannitol dehydrogenase in celery plants and cells.

Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.

A rapid, quantitative microplate assay for NAD-linked D-mannitol dehydrogenase.

A 96-well microtitre plate assay for NAD-linked D-mannitol dehydrogenase based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by reduced NAD is described. The assay allows rapid measurement of D-mannitol dehydrogenase in crude bacterial extracts derived by sonic disruption, in acetone permeabilized cells and in column eluates during enzyme purification. The absorbance of reaction mixtures in a microtitre plate is measured at 620 nm over a 3-4 min period using a programmable microplate reader. The rate of increase in absorbance is directly proportional to the amount of enzyme present and there is excellent correlation between activities derived using the microplate assay with those determined using conventional spectrophotometric methods.