Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.     

Biotechnological production of mannitol and its applications

Mannitol, a naturally occurring polyol (sugar alcohol), is widely used in the food, pharmaceutical, medical, and chemical industries. The production of mannitol by fermentation has become attractive because of the problems associated with its production chemically. A number of homo- and heterofermentative lactic acid bacteria (LAB), yeasts, and filamentous fungi are known to produce mannitol. In particular, several heterofermentative LAB are excellent producers of mannitol from fructose. These bacteria convert fructose to mannitol with 100% yields from a mixture of glucose and fructose (1:2). Glucose is converted to lactic acid and acetic acid, and fructose is converted to mannitol. The enzyme responsible for conversion of fructose to mannitol is NADPH- or NADH-dependent mannitol dehydrogenase (MDH). Fructose can also be converted to mannitol by using MDH in the presence of the cofactor NADPH or NADH. A two enzyme system can be used for cofactor regeneration with simultaneous conversion of two substrates into two products. Mannitol at 180 g l⁻¹ can be crystallized out from the fermentation broth by cooling crystallization. This paper reviews progress to date in the production of mannitol by fermentation and using enzyme technology, downstream processing, and applications of mannitol.     

Controlling substrate concentration in fed-batch candida magnoliae culture increases mannitol production

Candida magnoliae HH-01, a yeast strain that is currently used for the industrial production of mannitol, has the highest mannitol production ever reported for a mannitol-producing microorganism. However, when the fructose concentration exceeds 150 g/L, the volumetric mannitol production rate decreases because of a lag in mannitol production, and the yield decreases as a result of the formation of side products. In fed-batch culture, the volumetric production rate and mannitol yield from fructose vary substantially with the fructose concentration and are maximal at a controlled fructose concentration of 50 g/L. In continuous feeding experiments, the maximum mannitol yield was 85% (g/g) at a glucose/fructose feeding ratio of 1/20. A high glucose concentration in the production phase resulted in the formation of ethanol followed by a decrease in yield and productivity. NAD(P)H-dependent mannitol dehydrogenase was purified to homogeneity from C. magnoliae. In vitro, mannitol dehydrogenase was inhibited by increasing ethanol concentration. Mannitol product was also found to be inhibitory with a K(i) of 183 mM. Under optimum conditions, a final mannitol production of 213 g/L was obtained from 250 g fructose/L after 110 h.     

Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS(Mtl)). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Delta ldh Delta mtlA and Delta ldh Delta mtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo (13)C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS(Mtl). Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

Glucose catabolism in two derivatives of a Rhizobium japonicum strain differing in nitrogen-fixing efficiency.

Radiorespirometric and enzymatic analyses reveal that glucose-grown cells of Rhizobium japonicum isolates I-110 and L1-110, both derivatives of R. japonicum strain 3I1b110, possess an active tricarboxylic acid cycle and metabolize glucose by simultaneous operation of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. The hexose cycle may play a minor role in the dissimilation of glucose. Failure to detect the nicotinamide adenine dinucleotide phosphate-dependent decarboxylating 6-phosphogluconate dehydrogenase (EC 1.1.1.44) evidences absence of the pentose phosphate pathway. Transketolase and transaldolase reactions, however, enable R. japonicum to produce the precursors for purine and pyrimidine biosynthesis from fructose-6-phosphate and glyceraldehyde-3-phosphate. A constitutive nicotinamide adenine dinucleotide-linked 6-phosphogluconate dehydrogenase has been detected. The enzyme is stimulated by either mannitol or fuctose and might initiate a new catabolic pathway. R. japonicum isolate I-110, characterized by shorter generation times on glucose and greater nitrogen-fixing efficiency, oxidizes glucose more extensively than type L1-110 and utilizes preferentially the Embden-Meyerhof-Parnas pathway, whereas the Entner-Doudoroff pathway apparently predominates in type L1-110.     

Role of glucose in the bioconversion of fructose into mannitol by Candida magnoliae

The most efficient substrate for mannitol production by Candida magnoliae HH-01 is fructose; glucose and sucrose can also be converted into mannitol but with lower conversion yields. Mannitol dehydrogenase was purified and characterized; it had the highest activity with fructose as the substrate and used only NADPH. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration.     

The crystallographic structure of the mannitol 2-dehydrogenase NADP+ binary complex from Agaricus bisporus

Mannitol, an acyclic six-carbon polyol, is one of the most abundant sugar alcohols occurring in nature. In the button mushroom, Agaricus bisporus, it is synthesized from fructose by the enzyme mannitol 2-dehydrogenase (MtDH; EC ) using NADPH as a cofactor. Mannitol serves as the main storage carbon (up to 50% of the fruit body dry weight) and plays a critical role in growth, fruit body development, osmoregulation, and salt tolerance. Furthermore, mannitol dehydrogenases are being evaluated for commercial mannitol production as alternatives to the less efficient chemical reduction of fructose. Given the importance of mannitol metabolism and mannitol dehydrogenases, MtDH was cloned into the pET28 expression system and overexpressed in Escherichia coli. Kinetic and physicochemical properties of the recombinant enzyme are indistinguishable from the natural enzyme. The crystal structure of its binary complex with NADP was solved at 1.5-A resolution and refined to an R value of 19.3%. It shows MtDH to be a tetramer and a member of the short chain dehydrogenase/reductase family of enzymes. The catalytic residues forming the so-called catalytic triad can be assigned to Ser(149), Tyr(169), and Lys(173).     

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.     

Purification and characterization of mannitol dehydrogenase from Aspergillus parasiticus.

Mannitol dehydrogenase, NADP specific (EC 1.1.1.138), was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.4 X 10(5) and was composed of four subunits of apparently equal size. The substrate specificity was limited to D-mannitol, D-glucitol, D-arabinitol, 1-deoxy-D-mannitol, and 1-deoxy-D-glucitol. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to mannitol, with Ki and 1 microM. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of mannitol dehydrogenase.     

Fermentation of glucose, lactose, galactose, mannitol, and xylose by bifidobacteria

For six strains of Bifidobacterium bifidum (Lactobacillus bifidus), fermentation balances of glucose, lactose, galactose, mannitol, and xylose were determined. Products formed were acetate, l(+)-lactate, ethyl alcohol, and formate. l(+)-Lactate dehydrogenase of all strains studied was found to have an absolute requirement for fructose-1,6-diphosphate. The phosphoroclastic enzyme could not be demonstrated in cell-free extracts. Cell suspensions fermented pyruvate to equimolar amounts of acetate and formate. Alcohol dehydrogenase was shown in cell-free extracts. Possible explanations have been suggested for the differences in fermentation balances found for different strains and carbon sources. By enzyme determinations, it was shown that bifidobacteria convert mannitol to fructose-6-phosphate by an inducible polyol dehydrogenase and fructokinase. For one strain of B. bifidum, molar growth yields of glucose, lactose, galactose, and mannitol were determined. The mean value of Y (ATP), calculated from molar growth yields and fermentation balances, was 11.3.     

Enhanced nodulation of soybean by Bradyrhizobium in the presence ofPseudomonas fluorescens

Experiments were undertaken to determine the effect ofPseudomonas fluorescens on nodulation of soybean by two strains ofBradyrhizobium japonicum, USDA I-110 and 61A76.Pseudomonas fluorescens can enhance the nodulation ability ofB. japonicum. Preincubation ofB. japonicum withP. fluorescens before inoculation further increased the level of nodulation.     

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N₂-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K₂HPO₄-0.08% MgSO₄ · 7H₂O-0.02% NaCl-0.001% FeCl₃ · 6H₂O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.     

Fermentation of mannitol by Clostridium butyricum: role of acetate as an external hydrogen acceptor

Summary The continuous fermentation of mannitol (pH 6, dilution rate (D)=0.087 h^-1) by Clostridium butyricum LMG 1213t1 was investigated under several conditions. Mannitol was readily fermented when glucose or acetate were added in the in-flow medium as co-substrate. Butyrate, CO₂ and H₂ were the major fermentation products. In mannitol-glucose mixtures (ratios 4 or 8) the amount of mannitol fermented depended upon the amount of glucose in the in-flow medium. In mannitol-acetate mixtures, 1 mol of acetate was needed for the fermentation of approximately 5.5 mol mannitol. We detected d-mannitol-1-phosphate dehydrogenase activity, responsible for the generation of supplementary reduced nicotine adenine dinucleotide (NADH) as a source for extra H₂ gas. Fermentation of mannitol-acetate in the presence of [¹⁴C]-labelled acetate revealed butyrate as the only labelled fermentation end-product.     

Growth ofZymomonas mobilis CP4 on mannitol

Summary The ethanologenZymomonas mobilis has a restricted substrate range, namely glucose, fructose and sucrose. It would be useful to expand its substrate range to include other carbohydrates.Z. mobilis was screened for growth on 30 different carbohydrates and organic acids. A single spontaneous mutant,Z mobilis CP4.60, was isolated which illustrated feeble growth on mannitol as the sole carbohydrate source after three months of incubation. Growth ofZ. mobilis CP4.60 for several months in continuous culture with excess mannitol, and including a round of NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis in the chemostat, led to the isolation a sequential series of mutants (CP4.62, CP4.64 and CP4.66), each with improved growth rates on mannitol. Metabolism of mannitol byZ. mobilis is oxygen-dependent, resulting in limited production of ethanol and incresed production of lactic acid. This is an initial example of extension of the substrate range ofZymomonas. The conversion of mannitol to fructose could be via an altered alcohol dehydrogenase.     

Decoding the mannitol enigma in filamentous fungi

Mannitol is a 6-carbon polyol that is among the most abundant biochemical compounds in the biosphere. Mannitol has been ascribed a multitude of roles in filamentous fungi including carbohydrate storage, reservoir of reducing power, stress tolerance and spore dislodgement and/or dispersal. The advancement of genetic manipulation techniques in filamentous fungi has rapidly accelerated our understanding of the roles and metabolism of mannitol. The targeted deletion of genes encoding proteins of mannitol metabolism in several fungi, including phytopathogens, has proven that the metabolism of mannitol does not exist as a cycle and that many of the postulated roles are unsupported. These recent studies have provided a much needed focus on this mysterious metabolite and make this a fitting time to review the roles and metabolism of mannitol in filamentous fungi.     

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures.

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 mumol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecylsulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immuno-reactive 40-kD protein.     

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.)

In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in ¹⁴CO₂ labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bisphosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.