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.
Xylitol is a five-carbon sugar alcohol, i.e. polyol that has beneficial health properties. It prevents dental caries and acute otitis media (ear infection) in small children (Mäkinen 1992; Uhari et al. 1996). Xylitol can be found in nature in small quantities, for example in fruits and vegetables. Industrially, it is produced by the chemical hydrogenation of d-xylose into xylitol by metal catalyst. Xylitol has almost the same sweetness as sucrose, but with an energy value of only 2.4 cal/g compared to the 4 cal/g of sucrose. Xylitol has a negative heat of solution and good solubility in water, causing a cooling sensation when consumed orally. Due to its beneficial health effects, xylitol has become a global sweetener. It is mainly used as a sweetener in chewing gums, mints, sweets and toothpaste.
Background of xylitol production
The interest in xylitol production was originally created by its applicability to be used as a sweetener for diabetic people (Mellinghoff 1961; Lang 1964). The chemical and microbial production of xylitol was reported by Lohman (1957) and Onishi and Suzuki (1966, 1969). At that time, however, xylitol production from d-glucose was studied, as d-xylose was an expensive substrate. In 1970, an industrial scale chromatographic method for separating the different wood hemicellulose sugars was developed in Finland. This enabled the mass production of pure d-xylose. The method was developed where d-xylose was subsequently reduced to xylitol by a metal catalyst under high hydrogen pressure (Melaja et al. 1981; Härkönen and Nuojua 1979). At the same time, the beneficial effects of xylitol in preventing dental caries were found in Finland. The early research and development work of xylitol has been thoroughly reviewed by Mäkinen (2000).
Microbial production of xylitol with metabolically engineered Saccharomyces cerevisiae
Bruinenberg et al. (1983) studied the effect of different carbon and nitrogen sources on nicotinamide adenine dinucleotide phosphate (NADPH) consumption and production in Candida utilis. They concluded that during xylose metabolism, the main source of NADPH is the hexose monophosphate pathway and the isocitrate dehydrogenase enzyme. Few years later, it was suggested that redox imbalance between NADPH-dependent xylose reductase and NAD-dependent xylitol dehydrogenase was the main reason for xylitol accumulation (Bruinenberg 1986; van Dijken and Scheffers 1986; Rizzi et al. 1989). Kötter et al. (1990) constructed a xylose utilizing Saccharomycesscerevisiae strain. The next year, Hallborn et al. (1991) reported that they have succeeded in obtaining 95% conversion yield from xylose to xylitol with transformed S. cerevisiae strain. Kötter and Ciriacy (1993) suggested that the NADPH deficiency is the bottleneck of xylose fermentation in metabolically engineered S. cerevisiae. Consequently, it was assumed that the pentose phosphate pathway (PPP) in S. cerevisiae cannot regenerate a sufficient amount of NADPH during xylose metabolism under oxygen-limited conditions (Hallborn et al. 1994). Since then, the role of NADPH in xylose metabolism in yeasts has been studied intensively until today.
Different strategies have been studied to increase the NADPH generation in S. cerevisiae. Gancedo and Lagunas (1973) suggested that the flux into PPP is only 1–5% of the total carbon flux based on the labelled glucose measurements, although PPP is the main generator of NADPH in yeast metabolism. Lately, advanced methods have been applied, where flux into PPP and consequent NADPH regeneration has been quantified during glucose metabolism with different strains (Gombert et al. 2001; Fiaux et al. 2003). These results suggest that the PPP flux in S. cerevisiae can be up to 44/100 of consumed glucose molecules (Gombert et al. 2001). During glucose metabolism, the NADPH requirement is fulfilled by increased intracellular PPP flux in S. cerevisiae. However, during xylose metabolism, the PPP flux is not increased according to the requirements. Therefore, alternative strategies have been used, such as over-expressing the isocitrate dehydrogenase enzyme (Minard et al. 1998) or the newly discovered enzyme NADP-dependent d-glyceraldehyde 3-phosphate dehydrogenase (Verho et al. 2003), the metabolic engineering of the NADPH-generating malic enzyme in S. cerevisae (dos Santos et al. 2004) or over-expressing the endogenous NADPH-dependent aldose reductase (Traff-Bjerre et al. 2004). The recent advancements include removing the reduction–oxidation pathway of xylose into xylitol and replacing it with direct isomerization of d-xylose into d-xylulose (Kuyper et al. 2003). This approach removes the bottleneck in NADPH generation, allowing the NADPH-independent uptake of d-xylose and consequent fermentation to ethanol. The next susceptible bottleneck is the ATP-dependent uptake of d-xylulose, but it can possibly be alleviated by multi-expression of non-oxidative PPP enzymes (Johansson and Hahn-Hagerdal 2002). The uptake of d-xylose has been improved by spontaneous mutation techniques where genetically engineered S. cerevisiae cells have been adjusted to new growth conditions using different selection methods (Wahlbom et al. 2003; Sonderegger and Sauer 2003; Kuyper et al. 2005).
The main challenges concerning the use of metabolically engineered S. cerevisiae for industrial xylitol production are uptake of d-xylose and NADPH regeneration through PPP. The most definite way to generate a surplus of NADPH for intracellular reactions is to enhance the PPP flux. This far, the only definite way to increase PPP flux is to increase the growth rate of metabolically engineered S. cerevisiae during glucose metabolism (Gombert et al. 2001; Fiaux et al. 2003). However, as d-xylose is a non-preferred carbon substrate for S. cerevisiae, it does not provide sufficient amount of energy for growth and metabolism (Sonderegger et al. 2004). Therefore, it is assumed that d-xylose does not induce enhanced recirculation of carbon flux through PPP. In addition, alternative ways of increasing NADPH turnover by intracellular substrate cycling do not seem to have sufficient flux rate due to constraints in intracellular metabolism. Conversely, the d-xylose uptake has been improved by the combined use of isomerization and spontaneous mutation techniques in metabolically engineered S. cerevisiae. The produced d-xylulose can be directed to PPP through xylulokinase enzyme or converted back to xylitol by xylitol dehydrogenase enzyme. This opens up new possibilities considering bioethanol or even xylitol production with metabolically engineered S. cerevisiae.
Microbial production of xylitol with Candida yeasts
As previously mentioned, the interest in the role of NADPH in d-xylose metabolism began from the studies of Bruinenberg et al. (1983), although the research was later on directed to metabolically engineered S. cerevisiae (Kötter and Ciriacy 1993). Apparently, this was due to the long traditions in applying S. cerevisiae and the amount of scientific data already obtained from the baker’s yeast physiology. The research of xylitol production with Candida yeasts has mainly focused on optimizing the xylitol production conditions and the effect of oxygen. Metabolic engineering methods have not been applied extensively, as these methods have not been available for the Candida strains used in industrial applications. Only recently, there have been some reports on metabolic engineering of C. tropicalis related to xylitol production (Ko et al. 2006; Lee et al. 2003).
The oxygen availability is the most important factor in terms of xylitol production from d-xylose with Candida yeasts. Under oxygen limited conditions, the oxidative phosphorylation is not able to reoxidize all the generated NADH. Therefore, the intracellular concentration of NADH is increasing, resulting in xylitol accumulation. This mechanism was confirmed in controlled continuous culture conditions. Initially, descending agitation gradient was established in the bioreactor. It resulted in a gradual decrease in dissolved oxygen concentration and the onset of xylitol accumulation in C. guilliermondii (Granström et al. 2001) and C. tropicalis (Granström and Leisola 2002).
The d-xylose catabolic pathway consists of three enzymes: i.e. xylitol reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK). First, d-xylose is reduced to xylitol by XR and then oxidized to d-xylulose by XDH. Finally, d-xylulose is phosphorylated to d-xylulose 5-phosphate by XK, which then enters into PPP (Fig. 1). This reaction uses ATP and, therefore, the reaction rate is dependent on the energy charge and the phosphorylation potential of the cell. There is not an extensive scientific data available on the action of xylulokinase enzyme from Candida strains. However, it can be reasonably assumed that its function is similar to that in S. cerevisiae.
In C. tropicalis, the XR has a dual dependency for NADP(H), whereas in C. guilliermondii, the XR is strictly NADPH-dependent. The XDH is predominantly NAD-dependent, although an NADP-dependent XDH has also been reported by Rizzi et al. (1989). In terms of different cofactor dependency of XR, the C. tropicalis has a higher growth rate (D max) than C. guilliermondii in fully aerobic conditions on mineral medium in the carbon-limited chemostat. The maximum specific uptake rate and dilution rate for C. tropicalis and C. guilliermondii is 0.70 and 0.68 g g−1 cdw1 h−1 and 0.35 and 0.39 h−1, respectively (unpublished result). Both strains have been reported to have a very high yield of xylitol from d-xylose under oxygen limited conditions ranging from 0.78–0.82 (Ojamo 1994; Yahashi et al. 1996). However, there is a difference in their response to oxygen-limited conditions and the regeneration of cofactors.
In our controlled experimental set-up, the oxygen level was gradually decreased, and growth rate was increased within 10-h time in the chemostat. C. guilliermondii, whose XR is exclusively NADPH-dependent, accumulated acetate to regenerate NADPH, whereas C. tropicalis, whose XR is partly NADH-dependent, did not accumulate acetate. Both strains regenerated NAD by accumulation of glycerol, which is a known redox sink in S. cerevisiae (Oura 1997). The NAD regeneration was even more pronounced in C. tropicalis because it produced both glycerol and ethanol in these conditions. The different responses to oxidative stress caused by increased intracellular NADH concentration were further on studied by formate co-feeding. A fully aerobic steady state was established with no xylitol accumulation. Consequently, formate was fed as a cosubstrate, resulting in increased carbon dioxide production and intracellular NADH concentration by the action of formate dehydrogenase (Fig. 1). C. guilliermondii produced only glycerol, whereas C. tropicalis produced glycerol, ethanol and xylitol (Granström and Leisola 2002).
In case of C. guilliermondii, acetate accumulation was detected, as opposed to C. tropicalis in the chemostat conditions. The in vivo enzyme activities indicated that intracellular carbon fluxes recirculate through PPP to generate a sufficient amount of NADPH with simultaneous production by NADPH-dependent isocitrate dehydrogenase enzyme in C. guilliermondii (Granström et al. 2001). According to the metabolic flux analysis which was constructed on the basis of balancing the consumption and production of cofactors and energy, the PPP flux has to be at least 54% of the TCA flux to supply sufficient amount of NADPH for cell metabolism (unpublished result). Therefore, the extracellular acetate production could indicate a limitation in the PPP flux of C. guilliermondii, similarly to S. cerevisiae. C. tropicalis did not accumulate acetate, indicating that there was no limitation on NADPH, but NAD was regenerated by two different mechanisms, i.e. ethanol and glycerol production.
Considering the NAD regeneration, we need to look at the enzyme mechanism to interpret this result. The NAD-dependent XDH from xylose assimilating yeast Galactocandida mastotermitis was purified and the enzyme mechanism studied (Lunzer et al. 1998) and expressed in E.coli (Nidetzky et al. 2003). The K m and K i values for xylitol–NAD and d-xylulose–NADH were determined to be 39 and 410 μM and 10 and 27 μM, respectively, depending on the used buffer. They concluded that the enzyme reaction follows the mechanism where coenzyme binds first and leaves last. Therefore, dissociation of d-xylulose–NADH complex was suggested to be the rate-limiting step. In particular, d-xylulose was a strong competitive inhibitor with an inhibitor constant of 10 μM. The d-xylulose inhibition, in respect to NAD, was partly alleviated by increasing sorbitol concentration below saturating concentrations. When sorbitol concentration was increased, NADH gave non-competitive inhibition pattern at high concentrations of NAD. However, they suggested that the inhibition could probably be overcome by saturation of NAD (Lunzer et al. 1998).
According to these results, it can be concluded that both strains, i.e. C. guilliermondii and C. tropicalis, are accumulating xylitol in oxygen-limited conditions due to the increased NADH concentration and subsequent rate-limiting dissociation of d-xylulose–NADH complex (Rizzi et al. 1989). In addition, glycerol and ethanol accumulation indicates the necessity of NAD regeneration in both strains. This is due to the fact that most of the used substrates and cofactors exhibited competitive or mixed-type inhibition toward XDH. Consequently, NAD is regenerated to overcome the non-competitive inhibition by increased NADH, xylitol and d-xylulose concentrations. The substrate–product and NADH–NAD balance in respect to each other will determine the reaction rate and the direction of xylitol dehydrogenase enzyme.
The challenges in xylitol production using metabolically engineered S. cerevisiae have been thoroughly described in the literature. The uptake of glucose will induce the regeneration of NADPH through PPP, however, when d-xylose serves as a carbon substrate, this does not occur. This is due to the fact that d-xylose is a non-preferred carbon substrate for S. cerevisiae. Recently, encouraging results have been reported by improving the d-xylose uptake through spontaneous mutation techniques, opening new possibilities considering the bioethanol production. In terms of microbial production of xylitol, the most prominent production strain should have dual dependent XR, as it will partly abolish the bottleneck in NADPH regeneration through PPP. In terms of xylitol accumulation, the inhibition of XDH enzyme activity by intracellular NADH concentration and NAD regeneration are both equally significant biochemical reactions. In addition, the production strain should be a natural d-xylose consumer or possesses the corresponding characteristics in terms of d-xylose uptake and maintenance of the intracellular redox balance. Most of the Candida strains will fall into this category.
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Granström, T.B., Izumori, K. & Leisola, M. A rare sugar xylitol. Part I: the biochemistry and biosynthesis of xylitol. Appl Microbiol Biotechnol 74, 277–281 (2007). https://doi.org/10.1007/s00253-006-0761-3
- Pentose Phosphate Pathway
- Xylitol Production
- Xylose Metabolism