Production of arabitol from enzymatic hydrolysate of soybean flour by Debaryomyces hansenii fermentation

Arabitol is a low-calorie sugar alcohol with anti-cariogenic properties. Enzymatic hydrolysate of soybean flour is a new renewable biorefinery feedstock containing hexose, pentose, and organic nitrogen sources. Arabitol production by Debaryomyces hansenii using soybean flour hydrolysate was investigated. Effects of medium composition, operating conditions, and culture stage (growing or stationary phase) were studied. Production was also compared at different culture volumes to understand the effect of dissolved oxygen concentration (DO). Main factors examined for medium composition effects were the carbon to nitrogen concentration ratio (C/N), inorganic (ammonium) to organic nitrogen ratio (I/O-N), and sugar composition. Arabitol yield increased with increasing C/N ratio and a high I/O-N (0.8–1.0), suggesting higher yield at stationary phase of low pH (3.5–4.5). Catabolite repression was observed, with the following order of consumption: glucose > fructose > galactose > xylose > arabinose. Arabitol production also favored hexoses and, among hexoses, glucose. DO condition was of critical importance to arabitol production and cell metabolism. The yeast consumed pentoses (xylose and arabinose) only at more favorable DO conditions. Finally, arabitol was produced in fermentors using mixed hydrolysates of soy flour and hulls. The process gave an arabitol yield of 54%, volumetric productivity of 0.90 g/L-h, and specific productivity of 0.031 g/g-h.

     

D-Arabitol Production by Endomycopsis chodati

Endomycopsis chodati in an aerated fermentation produced d-arabitol in yields of 35 to 40% of the sugar supplied. Glucose, mannose, and sucrose were suitable substrates. A synthetic medium was developed for the fermentation that showed that nitrogen in the medium must be limiting to obtain high yields of arabitol. Excess phosphate also tended to lower arabitol yields, although the effect was not so great as with nitrogen. Pilot plant-size fermentations were made in which all the nutrients were supplied by blackstrap molasses and urea. Arabitol yields in these fermentations were about 40% of the sugar supplied.     

Nutritional and temporal control of arabitol and mannitol accumulation in Geotrichum

Summary Intracellular arabitol and mannitol accumulation is under nutritional and temporal control during arthrospore germination, vegetative growth, and arthrospore formation in Geotrichum. Arabitol is not produced if the glucose concentration in the medium is low. Arabitol is produced in large quantities in the cells if the carbon source is acetate or if the glucose level is above 10%. Low levels of glucose do not repress acetate induction of arabitol formation. Arabitol began to accumulate during spore swelling and vegetative growth in the presence of acetate. Mannitol appeared to serve as a carbon and energy reserve during starvation and arthrospore germination; the concentration of mannitol in vegetative cells remained barely detectable until sporulation commenced.This research was supported by National Science Foundation Grant GB-8327 and Public Health Service Grant Al-04603-09 to D.J.N.     

Production of arabitol from glycerol: strain screening and study of factors affecting production yield

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.     

Chemical and physiological changes in filamentous fungi during autolysis. X. Changes in the concentration of polyol in autolysing cultures of several fungi

Botrytis cinerea, Colletotrichum gloeosporioides, Aspergillus niger andPenicillium griseofulvum were grown in flasks as stationary cultures in the ordinary Czapek-Dox medium at 24° C in the dark for long periods of time. At convenient intervals during autolysis samples of mycelium were harvested and its polyol content determined. The loss of mycelial mannitol during autolysis reached to a little more than 60 %, more than 70 %, to nearly 80 % and 90 % forC. gloeosporioides, P. griseofulvum, A. niger andB. cinerea, respectively. Seventy per cent of the arabitol contained inP. griseofulvum and 85 % of the arabitol inC. gloeosporioides disappeared during autolysis. Mannitol remained present in appreciable amounts in these four fungi throughout the whole period of autolysis described, whereas arabitol disappeared fromB. cinerea and fromA. niger during the process.

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Zusammenfassung Botrytis cinerea, Colletotrichum gloeosporioides, Aspergillus niger undPenicillium griseofulvum sind als stationäre Kulturen in Czapek-Dox Nährboden bei 24° C lange Zeit in der Dunkelheit gezüchtet worden. In geeigneten Zwischenzeiten sind während der Autolyse Myzeliumproben entnommen und ihr Polyol-Inhalt bestimmt. Der Verlust vom myzelialen Mannitol während der Autolyse erreichte mehr als 60 %, mehr als 70 %, beinahe 80 % und 90 % inC. gloeosporioides, P. griseofulvum, A. niger, undB. cinerea, beziehungsweise. Siebzig Prozent von Arabitol inP. griseofulvum und 85 % von Arabitol inC. gloeosporioides verschwanden während der Autolyse. Mannitol verblieb in beträchtlicher Menge in diesen vier Pilzen während der ganzen Periode der Autolyse, während Arabitol vonB. cinerea und vonA. niger in derselben Zeit verschwand.

     

insilico Characterization and Homology Modeling of Arabitol Dehydrogenase (ArDH) from Candida albican

Background: Arabitol dehydrogenase (ArDH) is involved in the production of different sugar alcohols like arabitol, sorbitol, mannitol, erythritol and xylitol by using five carbon sugars as substrate. Arabinose, d-ribose, d-ribulose, xylose and d-xylulose are known substrate of this enzyme. ArDH is mainly produced by osmophilic fungi for the conversion of ribulose to arabitol under stress conditions. Recently this enzyme has been used by various industries for the production of pharmaceutically important sugar alcohols form cheap source than glucose. But the information at structure level as well as its binding energy analysis with different substrates was missing. Results: The present study was focused on sequence analysis, insilico characterization and substrate binding analysis of ArDH from a fungus specie candida albican. Sequence analysis and physicochemical properties showed that this protein is highly stable, negatively charged and having more hydrophilic regions, these properties made this enzyme to bind with number of five carbon sugars as substrate. The predicted 3D model will helpful for further structure based studies. Docking analysis provided free energies of binding of each substrate from a best pose as arabinose -9.8224calK/mol, dribose -11.3701Kcal/mol, d-ribulose -8.9230Kcal/mol, xylose -9.7007Kcal/mol and d-xylulose 9.7802Kcal/mol. Conclusion: Our study provided insight information of structure and interactions of ArDH with its substrate. These results obtained from this study clearly indicate that d-ribose is best substrate for ArDH for the production of sugar alcohols. This information will be helpful for better usage of this enzyme for hyper-production of sugar alcohols by different industries.     

Production of arabitol by yeasts: current status and future prospects

Arabitol belongs to the pentitol family and is used in the food industry as a sweetener and in the production of human therapeutics as an anticariogenic agent and an adipose tissue reducer. It can also be utilized as a substrate for chemical products such as arabinoic and xylonic acids, propylene, ethylene glycol, xylitol and others. It is included on the list of 12 building block C3‐C6 compounds, designated for further biotechnological research. This polyol can be produced by yeasts in the processes of bioconversion or biotransformation of waste materials from agriculture, the forest industry (l ‐arabinose, glucose) and the biodiesel industry (glycerol). The present review discusses research on native yeasts from the genera Candida, Pichia, Debaryomyces and Zygosaccharomyces as well as genetically modified strains of Saccharomyces cerevisiae which are able to utilize biomass hydrolysates to effectively produce l ‐ or d ‐arabitol. The metabolic pathways of these yeasts leading from sugars and glycerol to arabitol are presented. Although the number of reports concerning microbial production of arabitol is rather limited, the research on this topic has been growing for the last several years, with researchers looking for new micro‐organisms, substrates and technologies.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Glycerol and arabitol production by an intergeneric hybrid,PB2, obtained by protoplast fusion between Saccharomyces cerevisiae and Torulaspora delbrueckii

An intergeneric osmotolerant hybrid yeast, PB2, was used together with the parental strains to study glycerol and arabitol production in batch culture. This fusion product was previously obtained by protoplast fusion between Torulaspora delbrueckii and Saccharomyces cerevisiae. Polyols and biomass production were determined in batch culture under aerobic conditions. Under the conditions tested, using PB2 hybrid and both parental strains, the best results were obtained with the hybrid. Arabitol reached a final concentration of 70 g/l and glycerol was increased to up to 50 g/l. Electronic Publication     

Carbohydrate Composition and Acid Invertase Activity in Rice Leaves Infected with Pyricularia oryzae

Ethanol-soluble carbohydrates in healthy and blast-infected leaves and also cultures of Pyricularia oryzae were analyzed using gas chromatography. Arabitol, mannitol, and trehalose occurred in infected leaf tissue, but not in healthy controls. Cultures of P. oryzae contained mannitol and trehalose, but not arabitol and sucrose. The presence of arabitol only in blast-infected rice leaves suggests that arabitol synthesis may be induced in infected leaf tissue as a result of the rice-blast fungus interaction, but may not be within blast fungus itself. The amounts of glucose, fructose, and sucrose in infected leaves were slightly increased until 5 days, and greatly enhanced at 7 days after inoculation, There were no differences in amounts of these sugars between the cultivars Nakdong and Dobong. At 7 days after inoculation, increases in amounts of arabitol, mannitol, and trehalose were pronounced in the susceptible cultivar Nakdong than in the moderately susceptible cultivar Dobong. The increased amounts of glucose and fructose in infected plants of the two cultivars were closely correlated with the presence of a very active invertase.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

Non-antibiotic,efficient selection for alfalfa genetic engineering

A selectable marker gene (SMG), usually conferring resistance to an antibiotic or herbicide, is generally introduced into the plant cells with the gene(s) for the trait of interest to allow only the cells that have integrated and express the foreign sequences to regenerate into a plant. The availability of several SMGs for each plant species is useful for both basic and applied research to combine several genes of interest in the same plant. A selection system based on gabaculine (3-amino-2,3-dihydrobenzoic acid) as the selective substance and the bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde aminotransferase (GSA-AT)] as the SMG was previously used for genetic transformation of tobacco. The hemL gene is a good candidate for a safe SMG, because GSA-AT is present in all plants and is likely involved in one metabolic step only, so that unintended effects of its overexpression in plants are not probable. In this work, we have compared this new selection system with the conventional, kanamycin-based system for alfalfa Agrobacterium-mediated transformation. The hemL and NptII genes were placed together into a T-DNA under the control of identical promoters and terminators. We show that the gabaculine-based system is more efficient than the conventional, kanamycin-based system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect of its expression was observed.     

Characterisation and expression of monosaccharide transporters in lupins, <Emphasis Type="Italic">Lupinus polyphyllus</Emphasis> and <Emphasis Type="Italic">L. albus</Emphasis>

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

A metabolomic approach to dissecting osmotic stress in the wheat pathogen Stagonospora nodorum

A non-targeted metabolomics approach was used to identify significant changes in metabolism upon exposure of the wheat pathogen Stagonospora nodorum to 0.5M NaCl. The polyol arabitol, and to a lesser extent glycerol, was found to accumulate in response to the osmotic stress treatment. Amino acid synthesis was strongly down-regulated whilst mannitol levels were unaffected. A reverse genetic approach was undertaken to dissect the role of arabitol metabolism during salt stress. Strains of S. nodorum lacking a gene encoding an l-arabitol dehydrogenase (abd1), a xylitol dehydrogenase (xdh1) and a double-mutant lacking both genes (abd1xdh1) were exposed to salt and the intracellular metabolites analysed. Arabitol levels were significantly up-regulated upon salt stress in the xdh1 strains but were significantly lower than the wild-type. Arabitol was not significantly different in either the abd1 or the abd1xdh1 strains during osmotic stress but the concentration of glycerol was significantly higher indicating a compensatory mechanism in operation. Genome sequence analysis identified a second possible enzyme capable of synthesizing arabitol explaining the basal level of arabitol present in the abd1xdh1 strains. This study identified that arabitol is the primary compatible solute in S. nodorum but in-built levels of redundancy are present allowing the fungus to tolerate osmotic stress.     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol