Selenium stress response of the fruit origin strain Fructobacillus tropaeoli CRL 2034.

The fruit-origin strain Fructobacillus tropaeoli CRL 2034 can biotransform selenium into seleno-nanoparticles and selenocysteine. The proteomic analysis of F. tropaeoli CRL 2034 exposed to 5 and 100 ppm of Se showed a dose-dependent response since 19 and 77 proteins were deregulated, respectively. In the presence of 5 ppm of Se, the deregulated proteins mainly belonged to the categories of energy production and conversion or had unknown functions, while when cells were grown with 100 ppm of Se, most of the proteins were grouped into amino acid transport and metabolism, nucleotide transport and metabolism, or into unknown functions. However, under both Se conditions, glutathione reductases were overexpressed (1.8-3.1-fold), while mannitol 2-dehydrogenase was downregulated (0.54-0.19-fold), both enzymes related to oxidative stress functions. Mannitol 2-dehydrogenase was the only enzyme found that contained SeCys, and its activity was 1.27-fold increased after 5 ppm of Se exposure. Our results suggest that F. tropaeoli CRL 2034 counteracts Se stress by overexpressing proteins related to oxidative stress resistance and changing the membrane hydrophobicity, which may improve its survival under (food) storage and positively influence its adhesion to intestinal cells. Selenized cells of F. tropaeoli CRL 2034 could be used for producing Se-enriched fermented foods. KEY POINTS: • Selenized cells of F. tropaeoli showed enhanced resistance to oxidative stress. • SeCys was found in the Fructobacillus mannitol 2-dehydrogenase polypeptide chain. • F. tropaeoli mannitol 2-dehydrogenase activity was highest when exposed to selenium.

Identification of multiple proteins whose interaction with mannitol dehydrogenase is induced by salicylic acid: Implications for unconventional secretion.

Although protein secretion was previously believed to be solely via ER/Golgi pathways, Golgi-independent secretion has now been described in both animals and plants. Secretion of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in response to the endogenous pathogen response signal salicylic acid (SA) was one of the first reports of unconventional protein secretion in plants. To begin assessing potential secretion-associated MTD protein interactors, we present here high-quality databases describing changes in MTD-interacting proteins following SA treatment of Arabidopsis thaliana cells expressing MTD.

5-Keto-D-fructose production from sugar alcohol by isolated wild strain Gluconobacter frateurii CHM 43.

GLUCONOBACTER FRATEURII: CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.

MiRNAs differentially expressed in skeletal muscle of animals with divergent estimated breeding values for beef tenderness.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides, highly conserved among species, which modulate gene expression by cleaving messenger RNA target or inhibiting translation. MiRNAs are involved in the regulation of many processes including cell proliferation, differentiation, neurogenesis, angiogenesis, and apoptosis. Beef tenderness is an organoleptic characteristic of great influence in the acceptance of meat by consumers. Previous studies have shown that collagen level, marbling, apoptosis and proteolysis are among the many factors that affect beef tenderness. Considering that miRNAs can modulate gene expression, this study was designed to identify differentially expressed miRNAs that could be modulating biological processes involved with beef tenderness. RESULTS: Deep sequence analysis of miRNA libraries from longissimus thoracis muscle allowed the identification of 42 novel and 308 known miRNAs. Among the known miRNAs, seven were specifically expressed in skeletal muscle. Differential expression analysis between animals with high (H) and low (L) estimated breeding values for shear force (EBVSF) revealed bta-mir-182 and bta-mir-183 are up-regulated (q value < 0.05) in animals with L EBVSF, and bta-mir-338 is up-regulated in animals with H EBVSF. The number of bovine predicted targets for bta-mir-182, bta-mir-183 and bta-mir-338 were 811, 281 and 222, respectively, which correspond to 1204 unique target genes. Among these, four of them, MEF2C, MAP3K2, MTDH and TNRC6B were common targets of the three differentially expressed miRNAs. The functional analysis identified important pathways related to tenderness such as apoptosis and the calpain-calpastatin system. CONCLUSION: The results obtained indicate the importance of miRNAs in the regulatory mechanisms that influence muscle proteolysis and meat tenderness and contribute to our better understanding of the role of miRNAs in biological processes associated with beef tenderness.

Regulating Cofactor Balance In Vivo with a Synthetic Flavin Analogue.

A novel strategy to regulate cofactor balance in vivo for whole-cell biotransformation using a synthetic flavin analogue is reported. High efficiency, easy operation, and good applicability were observed for this system. Confocal laser scanning microscopy was employed to verify that the synthetic flavin analogue can directly permeate into Escherichia coli cells without modifying the cell membrane. This work provides a promising intracellular redox regulatory approach to construct more efficient cell factories.

Enhanced mannitol biosynthesis by the fruit origin strain Fructobacillus tropaeoli CRL 2034.

Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production and yield by the fruit origin strain Fructobacillus tropaeoli CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 22 full-factorial CCD with 4 axial points (α = 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l [95% CI (78.68, 91.39)] of mannitol and a yield of 82.02% [95% CI (71.98, 92.06)]. Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6-4.9 U/mg as the highest value found. To conclude, F. tropaeoli CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.

Genetically engineered Escherichia coli Nissle 1917 synbiotic counters fructose-induced metabolic syndrome and iron deficiency.

Consumption of fructose leads to metabolic syndrome, but it is also known to increase iron absorption. Present study investigates the effect of genetically modified Escherichia coli Nissle 1917 (EcN) synbiotic along with fructose on non-heme iron absorption. Charles foster rats weighing 150-200 g were fed with iron-deficient diet for 2 months. Probiotic treatment of EcN (pqq) and EcN (pqq-glf-mtlK) was given once per week, 109 cells after 2 months with fructose in drinking water. Iron levels, blood, and liver parameters for oxidative stress, hyperglycemia, and dyslipidemia were estimated. Transferrin-bound iron levels in the blood decreased significantly after 10 weeks of giving iron-deficient diet. Probiotic treatment of EcN (pqq-glf-mtlK) and fructose together led to the restoration of normal transferrin-bound iron levels and blood and hepatic antioxidant levels as compared to iron-deficient control group. The probiotic also led to the restoration of body weight along with levels of serum and hepatic lipid, blood glucose, and antioxidant in the blood and liver as compared to iron-deficient control group. Restoration of liver injury marker enzymes was also seen. Administration of EcN-producing PQQ and mannitol dehydrogenase enzyme together with fructose led to increase in the transferrin-bound iron levels in the blood and amelioration of consequences of metabolic syndrome caused due to fructose consumption.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.

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.

The mannitol utilization system of the marine bacterium Zobellia galactanivorans.

Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. In particular, mannitol can account for as much as 20 to 30% of the dry weight of brown algae and is likely to be an important source of carbon for marine heterotrophic bacteria. Zobellia galactanivorans (Flavobacteriia) is a model for the study of pathways involved in the degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and a fructokinase (FK). Among the observations, the M2DH of Z. galactanivorans was active as a monomer, did not require metal ions for catalysis, and featured a narrow substrate specificity. The FK characterized was active on fructose and mannose in the presence of a monocation, preferentially K(+). Furthermore, the genes coding for these two proteins were adjacent in the genome and were located directly downstream of three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis and showed the induction of these five genes after culture of Z. galactanivorans in the presence of mannitol as the sole source of carbon. This operon for mannitol catabolism was identified in only 6 genomes of Flavobacteriaceae among the 76 publicly available at the time of the analysis. It is not conserved in all Bacteroidetes; some species contain a predicted mannitol permease instead of a putative ABC transporter complex upstream of M2DH and FK ortholog genes.

Synthesis and anti-biofilm activities of dihydro-pyrrol-2-one derivatives on Pseudomonas aeruginosa.

Biofilm formation is an important reason for bacterial resistance to antimicrobials. Many compounds with dihydro-pyrrol-2-one (DPO) have antibacterial effects. It is prospective to base on DPO skeleton to design new compounds for biofilm inhibition. DPO was designed by a novel method of tandem cyclization between ethyl glyoxalate and amines, the series of DPO derivatives were synthesized by change of the amines. Their activities were evaluated by the inhibition of biofilm in Pseudomonas aeruginosa. The interaction of DPO derivatives with mannitol dehydrogenase (MDH) or extracellular DNA (eDNA) in the biofilm was simulated by molecular docking to reveal possible mechanism. 19 new DPO derivatives were synthesized and identified, 15 of them had antibacterial activities, but only 5 of them had more than 50% inhibition on biofilm of P. aeruginosa at 50µg/mL. The MDH activity and eDNA content in biofilm decreased significantly after treatment of the DPO derivatives in concentration dependence. The simulation reveals that strong interaction exists between the five DPO derivatives and MDH or eDNA, which are involved in anti-biofilm mechanism. The synthetic method of DPO derivatives is practical to provide effective anti-biofilm agents for P. aeruginosa, and they take effect through inhibition on MDH and eDNA of biofilm.

MTDH mediates trastuzumab resistance in HER2 positive breast cancer by decreasing PTEN expression through an NFκB-dependent pathway.

BACKGROUND: Trastuzumab resistance is almost inevitable in the management of human epidermal growth factor receptor (HER) 2 positive breast cancer, in which phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss is implicated. Since metadherin (MTDH) promotes malignant phenotype of breast cancer, we sought to define whether MTDH promotes trastuzumab resistance by decreasing PTEN expression through an NFκB-dependent pathway. METHODS: The correlations between MTDH and PTEN expressions were analyzed both in HER2 positive breast cancer tissues and trastuzumab resistant SK-BR-3 (SK-BR-3/R) cells. Gene manipulations of MTDH and PTEN levels by knockdown or overexpression were utilized to elucidate molecular mechanisms of MTDH and PTEN implication in trastuzumab resistance. For in vivo studies, SK-BR-3 and SK-BR-3/R cells and modified derivatives were inoculated into nude mice alone or under trastuzumab exposure. Tumor volumes, histological examinations as well as Ki67 and PTEN expressions were revealed. RESULTS: Elevated MTDH expression indicated poor clinical benefit, shortened progression free survival time, and was negatively correlated with PTEN level both in HER2 positive breast cancer patients and SK-BR-3/R cells. MTDH knockdown restored PTEN expression and trastuzumab sensitivity in SK-BR-3/R cells, while MTDH overexpression prevented SK-BR-3 cell death under trastuzumab exposure, probably through IκBα inhibition and nuclear translocation of p65 which subsequently decreased PTEN expression. Synergized effect of PTEN regulation were observed upon MTDH and p65 co-transfection. Forced PTEN expression in SK-BR-3/R cells restored trastuzumab sensitivity. Furthermore, decreased tumor volume and Ki67 level as well as increased PTEN expression were observed after MTDH knockdown in subcutaneous breast cancer xenografts from SK-BR-3/R cells, while the opposite effect were found in grafts from MTDH overexpressing SK-BR-3 cells. CONCLUSIONS: MTDH overexpression confers trastuzumab resistance in HER2 positive breast cancer. MTDH mediates trastuzumab resistance, at least in part, by PTEN inhibition through an NFκB-dependent pathway, which may be utilized as a promising therapeutic target for HER2 positive breast cancer.

Time-averaged distributions of solute and solvent motions: exploring proton wires of GFP and PfM2DH.

Proton translocation pathways of selected variants of the green fluorescent protein (GFP) and Pseudomonas fluorescens mannitol 2-dehydrogenase (PfM2DH) were investigated via an explicit solvent molecular dynamics-based analysis protocol that allows for direct quantitative relationship between a crystal structure and its time-averaged solute-solvent structure obtained from simulation. Our study of GFP is in good agreement with previous research suggesting that the proton released from the chromophore upon photoexcitation can diffuse through an extended internal hydrogen bonding network that allows for the proton to exit to bulk or be recaptured by the anionic chromophore. Conversely for PfM2DH, we identified the most probable ionization states of key residues along the proton escape channel from the catalytic site to bulk solvent, wherein the solute and high-density solvent crystal structures of binary and ternary complexes were properly reproduced. Furthermore, we proposed a plausible mechanism for this proton translocation process that is consistent with the state-dependent structural shifts observed in our analysis. The time-averaged structures generated from our analyses facilitate validation of MD simulation results and provide a comprehensive profile of the dynamic all-occupancy solvation network within and around a flexible solute, from which detailed hydrogen-bonding networks can be inferred. In this way, potential drawbacks arising from the elucidation of these networks by examination of static crystal structures or via alternate rigid-protein solvation analysis procedures can be overcome. Complementary studies aimed at the effective use of our methodology for alternate implementations (e.g., ligand design) are currently underway.

Coupling protein scaffold and biosilicification: A sustainable and recyclable approach for d-mannitol production via one-step purification and immobilization of multienzymes.

Enzymatic synthesis of biochemicals in vitro is vital in synthetic biology for its efficiency, minimal by-products, and easy product separation. However, challenges like enzyme preparation, stability, and reusability persist. Here, we introduced a protein scaffold and biosilicification coupled system, providing a singular process for the purification and immobilization of multiple enzymes. Using d-mannitol as a model, we initially constructed a self-assembling EE/KK protein scaffold for the co-immobilization of glucose dehydrogenase and mannitol dehydrogenase. Under an enzyme-to-scaffold ratio of 1:8, a d-mannitol yield of 0.692 mol/mol was achieved within 4 h, 2.16-fold higher than the free enzymes. The immobilized enzymes retained 70.9 % of the initial joint activity while the free ones diminished nearly to inactivity after 8 h. Furthermore, we incorporated the biosilicification peptide CotB into the EE/KK scaffold, inducing silica deposition, which enabled the one-step purification and immobilization process assisted by Spy/Snoop protein-peptide pairs. The coupled system demonstrated a comparable d-mannitol yield to that of EE/KK scaffold and 1.34-fold higher remaining activities after 36 h. Following 6 cycles of reaction, the immobilized system retained the capability to synthesize 56.4 % of the initial d-mannitol titer. The self-assembly co-immobilization platform offers an effective approach for enzymatic synthesis of d-mannitol and other biochemicals.

Dynamic mechanism of proton transfer in mannitol 2-dehydrogenase from Pseudomonas fluorescens: mobile GLU292 controls proton relay through a water channel that connects the active site with bulk solvent.

The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the "mobile" Glu(292), which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the "down → up" conformational change of Glu(292) could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu(292) → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu(292) as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu(292) is influenced by the bonding and charge state of the catalytic acid base Lys(295), suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD(+)-mannitol complex is promoted by a corresponding position change of Glu(292), which at physiological pH is associated with obligatory deprotonation of Lys(295) to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis.

Yarrowia lipolytica dehydrogenase/reductase: an enzyme tolerant for lipophilic compounds and carbohydrate substrates.

Yarrowia lipolytica short chain dehydrogenase/reductase (YlSDR) was expressed in Escherichia coli, purified and characterized in vitro. The substrate scope for YlSDR mediated oxidation was investigated with alcohols and unprotected carbohydrates spectrophotometrically, revealing a preference for secondary compared to primary alcohols. In reduction direction, YlSDR was highly active on ribulose and fructose, suggesting that the enzyme is a mannitol-2-dehydrogenase. In order to explore substrate tolerance especially for space-demanding, lipophilic protecting groups, 5-O-trityl-D-ribitol and 5-O-trityl-α,ß-D-ribose were investigated as substrates: YlSDR oxidized 5-O-trityl-D-ribitol and 5-O-trityl-α,ß-D-ribose and reduced the latter at the expense of NADP(H).

Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana.

Knockout and complement mutants of mannitol-1-phosphate dehydrogenase (MPD) and mannitol dehydrogenase (MTD) were constructed to probe the roles of both enzymes in the mannitol metabolism and multi-stress tolerances of entomopathogenic fungus Beauveria bassiana. Compared with wild-type and complement mutants, ΔBbMPD lost 99.5% MPD activity for reducing fructose-6-phosphate to mannitol-1-phosphate while ΔBbMTD lost 78.9% MTD activity for oxidizing mannitol to fructose. Consequently, mannitol contents in mycelia and conidia decreased 68% and 83% for ΔBbMPD, and 16% and 38% for ΔBbMTD, accompanied by greatly enhanced trehalose accumulations due to 81-87% decrease in their neutral trehalase expression. Mannitol as mere carbon source in a nitrate-based minimal medium suppressed the colony growth of ΔBbMTD instead of ΔBbMPD, and delayed more conidial germination of ΔBbMTD than ΔBbMPD. Based on median lethal responses, conidial tolerances to H(2) O(2) oxidation, UV-B irradiation and heat stress at 45°C decreased 38%, 39% and 22% in ΔBbMPD, and 18%, 16% and 11% in ΔBbMTD respectively. Moreover, ΔBbMPD and ΔBbMTD lost 14% and 7% of their virulence against Spodoptera litura larvae respectively. Our findings highlight the primary roles of MPD and MTD in mannitol metabolism and their significant contributions to multi-stress tolerances and virulence influential on the biocontrol potential of B.bassiana.

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source.

Mannitol is a natural polyol extensively used in the food industry as low-calorie sugar being applicable for diabetic food products. We aimed to evaluate mannitol production by Lactobacillus reuteri CRL 1101 using sugarcane molasses as low-cost energy source. Mannitol formation was studied in free-pH batch cultures using 3-10% (w/v) molasses concentrations at 37 °C and 30 °C under static and agitated conditions during 48 h. L. reuteri CRL 1101 grew well in all assayed media and heterofermentatively converted glucose into lactic and acetic acids and ethanol. Fructose was used as an alternative electron acceptor and reduced it to mannitol in all media assayed. Maximum mannitol concentrations of 177.7 ± 26.6 and 184.5 ± 22.5 mM were found using 7.5% and 10% molasses, respectively, at 37 °C after 24-h incubation. Increasing the molasses concentration from 7.5% up to 10% (w/v) and the fermentation period up to 48 h did not significantly improve mannitol production. In agitated cultures, high mannitol values (144.8 ± 39.7 mM) were attained at 8 h of fermentation as compared to static ones (5.6 ± 2.9 mM), the highest mannitol concentration value (211.3 ± 15.5 mM) being found after 24 h. Mannitol 2-dehydrogenase (MDH) activity was measured during growth in all fermentations assayed; the highest MDH values were obtained during the log growth phase, and no correlation between MDH activities and mannitol production was observed in the fermentations performed. L. reuteri CRL 1101 successfully produced mannitol from sugarcane molasses being a promising candidate for microbial mannitol synthesis using low-cost substrate.

[Efficient biosynthesis of D-mannitol by coordinated expression of a two-enzyme cascade].

D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.

Mannitol transport and mannitol dehydrogenase activities are coordinated in Olea europaea under salt and osmotic stresses.

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 µmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.