A rare sugar xylitol. Part I: the biochemistry and biosynthesis of xylitol

The rare sugar xylitol is a five-carbon polyol (pentitol) that has beneficial health effects. Xylitol has global markets and, therefore, it represents an alternative to current dominant sweeteners. The research on microbial reduction of d-xylose to xylitol has been focused on metabolically engineered Saccharomycess cerevisiae and Candida strains. The Candida strains have an advantage over the metabolically engineered S. cerevisiae in terms of d-xylose uptake and maintenance of the intracellular redox balance. Due to the current industrial scale production of xylitol, it has become an inexpensive starting material for the production of other rare sugar. The first part of this mini-review concentrates on the biochemistry of xylitol biosynthesis and the problems related to intracellular redox balance.     

Metabolic engineering strategies for improving xylitol production from hemicellulosic sugars

Xylitol is a five-carbon sugar alcohol with potential for use as a sweetener. Industrially, xylitol is currently produced by chemical hydrogenation of d-xylose using Raney nickel catalysts and this requires expensive separation and purification steps as well as high pressure and temperature that lead to environmental pollution. Highly efficient biotechnological production of xylitol using microorganisms is gaining more attention and has been proposed as an alternative process. Although the biotechnological method has not yet surpassed the advantages of chemical reduction in terms of yield and cost, various strategies offer promise for the biotechnological production of xylitol. In this review, the focus is on the most recent developments of the main metabolic engineering strategies for improving the production of xylitol.     

Evaluation of cotton stalk hydrolysate for xylitol production

Cotton stalk is a widely distributed and abundant lignocellulosic waste found in Turkey. Because of its rich xylose content, it can be a promising source for the production of xylitol. Xylitol can be produced by chemical or biotechnological methods. Because the biotechnological method is a simple process with great substrate specificity and low energy requirements, it is more of an economic alternative for the xylitol production. This study aimed to use cotton stalk for the production of xylitol with Candida tropicalis Kuen 1022. For this purpose, the combined effects of different oxygen concentration, inoculum level and substrate concentration were investigated to obtain high xylitol yield and volumetric xylitol production rate. Candida tropicalis Kuen 1022 afforded different concentrations of xylitol depending on xylose concentration, inoculum level, and oxygen concentration. The optimum xylose, yeast concentration, and airflow rate for cotton stalk hydrolysate were found as 10.41 g L^?1, 0.99 g L^?1, and 1.02 vvm, respectively, and under these conditions, xylitol yield and volumetric xylitol production rate were obtained as 36% and 0.06 g L^?1 hr^?1, respectively. The results of this study show that cotton stalk can serve as a potential renewable source for the production of xylitol.     

Covalent immobilization of recombinant <Emphasis Type="Italic">Rhizobium etli</Emphasis> CFN42 xylitol dehydrogenase onto modified silica nanoparticles

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t _1/2 of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K _m of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into l-xylulose. A ∼ 71% conversion and a productivity of 10.7 g h^-1 l^-1 were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to l-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.     

Selection and characterisation of a xylitol-derepressed Aspergillus niger mutant that is apparently impaired in xylitol transport

Aspergillus niger is known for its biotechnological applications, such as the use of xylanase enzyme for the degradation of hemicellulose. Depending on culture conditions, several polyols may also be accumulated, such as xylitol during D-xylose oxidation. Also during industrial fermentation of xylose for the production of fuel ethanol by recombinant yeast, xylitol is a by-product. We studied xylitol metabolism by isolating mutants that have impaired xylitol-mediated repression. Genetic and biochemical characterisation revealed that one of these mutants was affected not only in xylitol-mediated carbon repression, but also had impaired xylitol transport.The first two authors have contributed equally to this work.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.     

The production of xylitol by enzymatic hydrolysis of agricultural wastes

Agricultural waste products, beech wood and walnut shells, were hydrolyzed at 40°C using mixed crude enzymes produced byPenicillium sp. AHT-1 andRhizomucor pusillus HHT-1.d-xylose, 4.1 g and 15.1 g was produced from the hydrolysis of 100 g of beech wood and walnut shells, respectively. For xylitol production,Candida tropicalis IFO0618 and the waste product hydrolyzed solutions were used. The effects on xylitol production, of adding glucose as a NADPH source,d-xylose and yeast extract, were examined. Finally, a 50% yield of xylitol was obtained by using the beech wood hydrolyzed solution with the addition of 1% yeast extract and 1% glucose at an initial concentration.     

Screening of yeasts for production of xylitol fromd-xylose and some factors which affect xylitol yield inCandida guilliermondii

Summary The ability to convertd-xylose to xylitol was screened in 44 yeasts from five genera. All but two of the strains produced some xylitol with varying rates and yields. The best xylitol producers were localized largely in the speciesCandida guilliermondii andC. tropicalis. Factors affecting xylitol production by a selectedC. guilliermondii strain, FTI-20037, were investigated. The results showed that xylitol yield by this strain was affected by the nitrogen source. Yield was highest at 30–35°C, and could be increased with decreasing aeration rate. Using high cell density and a defined medium under aerobic conditions, xylitol yield byC. guilliermondii FTI-20037 from 104 g/ld-xylose was found to be 77.2 g/l. This represented a yield of 81% of the theoretical value, which was computed to be 0.9 mol xylitol per mold-xylose.Issued as NRCC publication No. 28798.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arabinose pathway engineering for arabitol-free xylitol production in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce l-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from l-arabinose to arabitol, the l-arabinose-preferring, endogenous XR was replaced by a d-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of l-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g l-arabinose l⁻¹ into 9.5 and 8.3 g arabitol l⁻¹, respectively, whereas the recombinant strain JY consumed 10.5 g l-arabinose l⁻¹ for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.     

Fermentation behavior of osmophilic yeast Candida tropicalis isolated from the nectar of Hibiscus rosa sinensis flowers for xylitol production

Eighteen yeast species belonging to seven genera were isolated from ten samples of nectar from Hibiscus rosa sinensis and investigated for xylitol production using d-xylose as sole carbon source. Amongst these isolates, no. 10 was selected as the best xylitol producer and identified as Candida tropicalis on the basis of morphological, biochemical and 26S rDNA sequencing. C. tropicalis produced 12.11 gl⁻¹ of xylitol in presence of 50 gl⁻¹ of xylose in 72 h at pH 5, 30°C and 200 rpm. The strain of C. tropicalis obtained through xylose enrichment technique has resulted in a yield of 0.5 gg⁻¹ with a xylitol volumetric productivity of 1.07 gl⁻¹h⁻¹ in the presence of 300 gl⁻¹ of xylose through batch fermentation. This organism has been reported for the first time from Hibiscus rosa sinensis flowers. Realizing, the importance of this high valued compound, as a sugar substitute, xylose enrichment technique was developed in order to utilize even higher concentrations of xylose as substrate for maximum xylitol production.     

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.     

Downstream processing for xylitol recovery from fermented sugar cane bagasse hydrolysate using aluminium polychloride

Xylitol, a sweetener comparable to sucrose, is anticariogenic and can be consumed by diabetics. This sugar has been employed successfully in many foods and pharmaceutical products. The discovery of microorganisms capable of converting xylose present in lignocellulosic biomass into xylitol offers the opportunity of producing this poliol in a simple way. Xylitol production by biotechnological means using sugar cane bagasse is under study in our laboratories, and fermentation parameters have already been established. However, the downstream processing for xylitol recovery is still a bottleneck on which there is only a few data available in the literature. The present study deals with xylitol recovery from fermented sugar cane bagasse hydrolysate using 5.2 g/l of aluminium polychloride associated with activated charcoal. The experiments were performed at pH 9, 50 degrees C for 50 min. The results showed that aluminium polychloride and activated charcoal promoted a 93.5% reduction in phenolic compounds and a 9.7% loss of xylitol from the fermented medium, which became more discoloured, facilitating the xylitol separation.     

Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62 g l⁻¹ h⁻¹, respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.     

Perspectives of engineering lactic acid bacteria for biotechnological polyol production

Polyols are sugar alcohols largely used as sweeteners and they are claimed to have several health-promoting effects (low-caloric, low-glycemic, low-insulinemic, anticariogenic, and prebiotic). While at present chemical synthesis is the only strategy able to assure the polyol market demand, the biotechnological production of polyols has been implemented in yeasts, fungi, and bacteria. Lactic acid bacteria (LAB) are a group of microorganisms particularly suited for polyol production as they display a fermentative metabolism associated with an important redox modulation and a limited biosynthetic capacity. In addition, LAB participate in food fermentation processes, where in situ production of polyols during fermentation may be useful in the development of novel functional foods. Here, we review the polyol production by LAB, focusing on metabolic engineering strategies aimed to redirect sugar fermentation pathways towards the synthesis of biotechnologically important sugar alcohols such as sorbitol, mannitol, and xylitol. Furthermore, possible approaches are presented for engineering new fermentation routes in LAB for production of arabitol, ribitol, and erythritol.     

Microbial production of xylitol from D-xylose using Candida tropicalis

Candida tropicalis DSM 7524 was used to produce xylitol from d-xylose. The fermentation conditions were optimized during continuous cultivation. The strain employed showed no great dependence upon temperature in a range between 30° C and 37° C. It achieved its best yield of xylitol from d-xylose at a pH value of 2.5. Such low pH values allow non sterile cultivation, which is a major economic factor. With an oxygen uptake rate of 0.8–1 ml oxygen per litre culture medium, the C. tropicalis produce xylitol at a yield of between 77% and 80% of the theoretical value. Higher yeast extract concentrations prevent the conversion of d-xylose into xylitol. d-xylose acts as a growth inhibitor in higher concentrations. The maximum xylitol yield was reached at a d-xylose concentration of around 100 g/l. In a non sterile batch culture with substrate shift 220 g/l xylitol were produced from 300 g/l d-xylose at a xylitol productivity rate of 0.37 g/(lh). In order to increase the specific yield, C. tropicalis was immobilised on porous glass and cultivated in a fluidized bed reactor. In a continuous non sterile cultivation with immobilised cells 155 g/l d-xylose produced 90–95% g/l xylitol with a productivity of 1.35 g/(lh).Mr. S. S. da Silva was a visiting scientist to the GBF. He was supported by a scholarship from the National Council of Scientific and Technological Development, Brasilia, Brazil (CNPq).We also would like to gratefully acknowledge the support of Prof. Dr. Michele Vitolo of the University of Sao Paulo, and the Centre for Biotechnology and Chemistry, Lorena, S. P. Brazil, in particular the Department of Fermentative Process.We are grateful to Prof. Rainer Jonas, head of the International Cooperation between Germany/Brazil for the helpful discussions and Dr. Heinrich Lönsdorf (GBF) for the Scanning electron micrographs.Dedicated to the 65th birthday of Prof. Dr. Fritz Wagner.     

d-Glucose feeding for improvement of xylitol productivity from d-xylose using Candida tropicalis immobilized on a non-woven fabric

Summary A non-woven fabric was successfully applied for immobilization of Candida tropicalis to produce xylitol from d-xylose. Xylitol productivity was enhanced by feeding of d-glucose (50 g/l·d); 87 g xylitol/L was produced after 64 h cultivation. Non-woven fabric could be used five times for fed-batch cultivation.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002