Romanian Biotechnological Letters Copyright 2010 University of Bucharest

Vol. 15, No.3, 2010 Printed in Romania. All rights reserved REVIEW

Production of xylitol by yeasts

Received for publication, September 17, 2009 Accepted, May 3, 2010 RALUCA GHINDEA1, ORTANSA CSUTAK2, ILEANA STOICA2, ANA-MARIA TANASE2, TATIANA VASSU2 1 MICROGEN Center for Research in Genetics, Microbiology and Biotechnology, University of Bucharest, Faculty of Biology, 1-3 Aleea Portocalilor, sector 5, 060101 Bucharest, Romania 2 University of Bucharest, Faculty of Biology, Department of Genetics, 1-3 Aleea Portocalilor, sector 5, 060101 Bucharest, Romania *E-mail: rghindea@gmail.com

Abstract
Xylitol is a naturally occuring sugar alcohol, which is currently produced chemically on a large scale. In recent years the bioconversion of D-xylose from lignocellulosic residues into xylitol, gained an increased attention as an alternative procedure , due to its high efficiency and reduced cost of such technology. The biotechnological method of producing xylitol involves microorganisms that are able to utilize xylose. Among them different yeast species had been proved to be very efficient in xylitol production. Our study deals with some aspects regarding xylose transport and metabolism within the yeast cell, and factors that can affect xylitol production. There are also methods for improving xylitol bioconversion.

Keywords: xylitol, xylose metabolism, yeasts

Introduction
Sugar alcohols are a class of polyols with applications in improvement of food nutritional characteristics. They present health advantages such as: lower caloric content, cancer fighting effects and lowering of the glycemic index. Also, sugar alcohols received special attention due to their applications in pharmaceuticals, animal nutrition and chemical production. They occur naturally in fruits and vegetables and can also be produced by microorganisms such as yeasts and bacteria. A polyol largely in use at present is xylitol [1]. Industrially, the polyols, including xylitol, are produced by chemical hydrogenation of sugars (such as D-xylose), which requires nickel catalysts, as well as high temperature and high pressure conditions. Since the costs associated with the traditional industrial production, as well as with the in vitro enzyme-based manufacturing are rather high, the biotechnological method of producing xylitol by microorganisms has received an increased interest. Over the years it was shown that this alternative is very specific and the microbial production can be maximized by metabolic engineering. Biotechnological production of xylitol Xylitol is a rare sugar that occurs naturally, albeit in small amounts, in fruits, vegetables, lichens, mushrooms, etc. Because the industrial scale production is possible [2] (Figure 1), it can be used as a raw material for the production of other rare sugars. For example it can be oxidized to L-xylulose by xylitol- dehydrogenase. The obtained compound can lead to L-lyxose or L-xylose in the presence of L-rhamnose isomerase enzyme. At the same time, L-xylulose can transform to L-ribulose and finally to L-arabinose by L-arabinose isomerase [3].

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Biotechnological production of xylitol was extensively studied as an alternative to the industrial one in order to clarify the metabolic pathways involved in microbial growth in the presence of non-conventional compounds [4,5,6](Table 1).
Table 1. Species acceptable for biotechnological production of xylitol Enterobacter liqufaciens Corynebacterium sp. Mycobacterium smegmatis Escherichia coli Saccharomyces cerevisiae Pichia stipitis Pichia farinosa Debaryomyces hansenii Pachysolen tannophilus Candida guilliermondii Candida tropicalis Candida boidinii Candida parapsilosis Candida pelliculosa
Xilose

Hansenula polymorpha Trichosporon sp. Rhodotorula sp. Cryptococcus sp. Kluyveromyces sp.

Fruits and vegetables

Lignocellulosics material (rich in xylan)

Extraction

Hydrolysis

Hylose-containing hydrolysate

Detoxification H2 Microorganisms Hydrogenation Biotehnological conversion

Separation/Purification

XYLITOL

Figure 1. Xylitol production methods [7]

Yeasts from genera Candida, Pichia, Debaryomyces, and Pachysolen are especially able to produce xylitol from D-xylulose, through successive metabolic reactions, with various yields [8]. Xylose transport within the yeast cell It was recently proved that Saccharomyces cerevisiae can grow slowly in the presence of xylose as sole carbon source, in aerobic conditions. Until recently, it was considered that S. cerevisiae does not present specific transporters for xylose and that grows poorly in its presence [9]. The initial studies regarding the xylose transport alternatives within the yeast cell involved strains of P. stipitis, P. heedii, C. shehatae, and C. intermedia and proved the existence of two transport systems. 1. A facilitated diffusion system, with low affinity the genes involved here are, for example, SUT1 (sugar transporter 1) for P. stipitis, or GXF1 (glucose/xylose facilitator 1) for C. intermedia, which are constitutively expressed genes that code the glucose/xylose transporter proteins [10] 2. A symport xylose-proton system, with high affinity - GXS1 (glucose/xylose symporter that codes proteins involved in the symport monosaccharides-protons transport in various species of yeasts and fungi.
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Production of xylitol by yeasts

H H OH H OH XYLOSE

O OH
H OH H H

Extracellular fluid

XHT

H+
symport

Cell membrane

Cytoplasm
XR XDH

XYLITOL
XI

XYLOSE

XYLULOSE

Figure 2. Xylose transport within the yeast cell (XHT- hexose transporters; XR-xylose-reductase; XDH-xylitol-dehydrogenase;XI xylose-isomerase) [11]

In the case of S. cerevisiae species, the facilitated diffusion of xylose takes place with the aid of transporter proteins coded by HXT (hexose transporter) genes (Figure 2). The xylose transport in the S. cerevisiae cell is, however, less efficient than glucose transport, the transport proteins (code by the genes XHT2, XHT6, XHT7) manifesting higher affinity for glucose. Xylose Metabolism in yeast cells In the first stage, D-xylose is transformed into an intermediary: xylitol, with the help of a xylose-reductase (XR), coded by XYL1, in the presence of NADH or NADPH. Afterwards, the xylitol is transformed into D-Xylulose by the xylitol- dehydrogenase (XDH), product of the XYL2 gene, which can use as co-factors NAD+ or NADP+ (Figure 3) [12,13]. Additionally, S. cerevisiae, can be made to produce xylitol from xylose by cloning the XYL1 gene from P. stipitis or C. tropicalis. It is well known that yeasts can present differences in their ability to ferment xylulose into ethanol : - in anaerobic conditions, as well as in the presence of low oxygen concentrations, yeasts that present the xylose-reductase with activity depending on NADH and/or NADPH (as for example P. stipitis) can regenerate NAD+ used in the second metabolic reaction. In this case the main product is ethanol and there is no xylitol accumulation. - in the case of yeast strains whose xylose-reductase cannot be used as a co-factor NADH, xylitol accumulation takes place in the first stage of the xylose metabolic pathway (example : Debaryomyces hansenii). The factors influencing the xylitol production in yeasts The fermentation process that produces xylitol in yeasts is controlled by a series of factors such as : substrate concentration, carbon source, inoculum, aeration degree, temperature or pH. 1. Xylose Concentration It was proved experimentally that one of the parameters influencing the yeast growth and the fermentation process is the substrate concentration (D-Xylose). The initial xylose concentration can influence the xylitol production. This way, in the case of microorganisms that can grow in high osmotic pressure conditions or in the presence of elevated glucides
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concentrations, a high initial xylose concentration can lead to a higher quantity of xylitol. At the same time as the initial concentration of xylose rises, it should take place an increase of the oxygen level, therefore avoiding the inhibition of the microbial growth. Experimental studies on C. tropicalis strains show that, at high xylose concentrations and in optimal aeration rate, a significant cellular growth takes place at the beginning of the fermentation process, and the xylitol production rate is considerably improved.

Figure 3. Xylose Metabolism in yeasts [14]

2. The Carbon Source It was proved experimentally that, in the case of C. tropicalis, the use of D-glucose as substrate in low concentration, also leads to a growth in production efficiency. This effect can be explained by the fact that the D-glucose is used in cell growth, D-xylose being consumed only afterwards. The influence of this factor also proved to be specific to certain species, since similar results have not been confirmed in other cases, such as C. guilliermondii [15]. 3. Nitrogen Source Among nitrogen sources, the yeast extract and the urea are the nutrients preferred by the yeasts producing xylitol. These nitrogen sources stimulation effect was proved by studies on some C. boidinii strains. 4. Concentration and inoculum age The fermentation process can be influenced also by the inoculum age, this affecting the metabolic activity and the viability of the cells. An amelioration of xylitol production by C. guilliermondii (0.75gl-1h-1), starting from a 24h inoculum and an initial xylose concentration of 54.5 g l-1 succeeded experimentally. 5. Aerobic Conditions Oxygen represents an important factor in xylose degradation by yeasts. It has been proved that in total anaerobic conditions, a complete stop of the metabolic pathway that produces xylitol from xylose takes place. The oxygen level needed for xylose metabolism is
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also a component specific to each species. For example, C. tropicalis shows a maximum of productivity in semi-anaerobic conditions, similar results being confirmed also when using D. hansenii strains ( 4-22 mmol l-1min-1). On the other hand, in the case of growth cultivation in the presence of glucose, aerobic conditions are necessary due to the high cellular density being obtained. 6. Temperature and pH Optimal temperature has been experimentally found to be around the value of 30C, with small variations not significantly affecting the xylitol concentration to be obtained. However, when the cells are grown at temperatures beyond 37C, a drastic decrease of productivity is observed. The initial pH value used during the fermentation process is being chosen depending on the species used. The optimal pH for D. hansenii is 5.5, while for C. parapsilosis, C. guilliemondii and C. boidinii the values are 4.5-5, 6.0, and 7.0 respectively. Methods to improve the xylitol production by yeasts The ability to obtain xylitol as a normal metabolic product was proved for different species of yeasts, especially belonging to Candida genera (C.boidinii, C.guilliermondii, C.parapsilosis,,C.pelliculosa, C.shehatae, C.tropicalis [16,17] and the related species Debaryomyces hansenii and Pichia stipitis. Although these yeasts are the most active and therefore the most useful in xylitol production, problems still rise at an industrial scale due to the high production cost and the high price for D-xylose. These can be surpassed by applying methods to optimize the production process, involving modifying the growth conditions, controlling the dissolved oxygen and the redox potential, using certain co-substrate or modifying the enzyme-related activity by blocking the expression of the XYL2 gene and thus amplifying the xylosereductase activity. The improvement methods described until now are based on: 1. Chemical (with ethylmetansulphonate-EMS) and physical (with ultraviolet radiation) mutagenesis techniques. The number of xylitol-producing mutants can be raised, in this case, by following with experiments with N-metil-N-nitro-N nitrosoguanidine (MNNG). The selection of mutants of interest is done, in both cases, by growing over specific media. 2. Genetic engineering techniques Until now, there have been described three categories of yeast strains that can produce xylitol. 1. Wild strains that can assimilate xylose with production of xylitol (for example: C. boidinii, C. guilliermondii, C. tropicalis, C.parapsilosis, D. hansenii) [18, 19]; 2. Yeast strains (for example : P.stipitis) that can convert xylose in xylitol due to the disruption at the level of the genes that code for alcohol-dehidrogenase (ADH), or xylitoldehidrogenase (XDH); 3. Recombined yeast strains such as S. cerevisiae that present the XYL1 gene from P. stipitis. (Ying Y.S., 2005) or C. tropicalis presenting the gene XYL1 from C. parapsilosis [20]. For instance Ko and his collaborators demonstrated recently [21,22] that the optimizing of xylitol production in C. tropicalis can be obtained by blocking the metabolic transformation of xylitol at D-xylulose as a result of genetic disruption at the level of the XYL2 gene. By adding a co-substrate (the most efficient one proved to be glycerol) necessary to cellular growth and NADPH regeneration, the final xylitol concentration obtained was 48.6 g/l in 16h.

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This strategy was also successfully used in the case of other yeast species such as P. stipitis. Defective mutants in XDH synthesis, obtained through the disruption of the XYL3 gene coding for D-xylulokinase, can lead to xylitol accumulation (26g/l) in media [23]. Acknowledgements We wish to thank P. Chipurici (POLITEHNICA University of Bucharest) and C. ChisegaNegrila (OVM-ICCPET SA) for kindly providing us valuable information and for helpful discussions. This study has supported by National Center for Programs Management CNMP (PNII 62-064) grant.

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