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Xylose, like glucose, is taken up into the yeast cell by facilitated diffusion (Kotyk 1967; Busturia and Lagunas 1986), predominantly by the action of hexose transporters, which are thought to transport the sugar concomitantly with glucose (Hamacher et al. 2002; Lee et al. 2002; Gardonyi et al. 2003a; Sedlak and Ho 2004). Kötter and Ciriacy (1993) varied the concentrations of the two sugars in a growth medium and showed that xylose was transported into the cell at a much lower rate than glucose because the transporters showed a 200-fold lower affinity for the pentose sugar than for the hexose. They determined the Km for xylose to be 190 mM to 1.5 M, leading to the suggestion that there are high-affinity and low-affinity systems for xylose uptake. This suggested the Km, when compared to that of glucose (1.5 mM to 35 mM), showed that the monosaccharide transport system had a 200-fold lower affinity for xylose than for glucose. Kötter and Ciriacy (1993) proposed that, in order for the cell to overcome the transport limitation, the xylose concentration in the medium should be higher than the Km but suggested the initial concentration of 133 mM (20 g/L) used in their study, did not limit xylose utilisation. Hamacher et al. (2002) studied the effect of each individual transporter during xylose transport by using a mutant strain with all the hexose transporters deleted (Wieczorke et al. 1999). They showed that HXT4, HXT5 and HXT7 were involved in xylose transport, with HXT7 being the most active. They found that the GAL2 gene coding for galactose permease (Kou et al. 1970) also had xylose- transporting ability. This correlated with the findings of Lindén et al. (1992), who reported that cells grown on galactose and xylose together had a better growth rate in a sulfite liquor plant. With the apparent similarities in structure, it is surprising to note that no further studies have been published in relation to the effect of galactose in xylose fermentation. Sedlak and Ho (2004) also carried out a characterisation study of hexose transporters during xylose utilisation and postulated that HXT5 was the major xylose transporter. Xylose transport in S. cerevisiae has been thought to be one of the limiting steps for fermentation, especially at low concentrations in the medium. Even though there are several limitations downstream (as discussed in the following sections), it might become crucial at a later stage to increase the transport rate of xylose in S. cerevisiae either by altering the native transporter or by cloning and expressing xylose-specific transporter(s) from other organisms.

Many natural xylose-utilising yeasts have been thought to possess transporters that can transport xylose, probably even at low concentrations (<1 g/L) (Alcorn and Griffin 1978; Lucas and Van Uden 1986; Kilian and Van Uden 1988; Does and Bisson 1989; Kilian et al. 1993; Nobre et al. 1999; Weierstall et al. 1999). While no xylose- specific transport gene has been isolated so far, the mechanism by which xylose transport occurs differs among organisms. For instance, P. stipitis is thought to transport xylose through two proton symport systems, one of high and the other of low affinity (Kilian and Van Uden 1988; Does and Bisson 1989). In most cases, two kinetically distinct xylose transporting systems have been reported (Fig. 2.3). The low-affinity transporter is generally shared with glucose and the high-affinity transporter is specific

for xylose. The low-affinity system transports xylose through facilitated diffusion, while the high-affinity system is thought to symport xylose with a proton using proton motive force. C. shehatae, when grown on either D-glucose or D-xylose, produced a facilitated

diffusion system that could transport D-glucose (high affinity/low capacity), D-xylose (low affinity/high capacity), or D-mannose (Lucas and Van Uden, 1986). Thus, all these three

FIG. 2.3 A, B. The two mechanisms of xylose uptake by yeast: A. facilitated diffusion – the concentration gradient is the driving force – these transporters have a broader substrate range; B. proton-xylose symport – driving force is the proton motive force, maintained by the plasma membrane proton-ATPase. Adapted from Weusthuis

et al. (1994).

sugars were competitive inhibitors of each other. Due to the high capacity of the system,

D-xylose was taken up even in the presence of D-glucose. Another transport system that

has been reported is that of the presence of glucose-repressible, high-affinity and constitutive facilitated diffusion xylose uptake systems in Candida intermedia (Gardonyi et al. 2003a; 2003b). Leandro et al. (2006) reported on the characterisation of the two transporters from C. intermedia. One was a facilitator for glucose/xylose called GXF1 (Km for xylose 0.4 mM), while the other was a glucose/xylose symporter named GXS1. It was thought that GXF1 would be more efficient under oxygen-limited or anaerobic fermentation due to its lower energy needs for activation in contrast to the symporter system. P. stipitis is supposed to possess three different transporters, each having different levels of affinities for the sugar. Three genes (SUT1, SUT2 and SUT3) were cloned and sequenced (Weierstall et al. 1999). SUT1 had a Km of 145 mM for xylose and seemed to be the major contributor to the low-affinity component of the glucose and xylose transport, which was evident from the lack of a low-affinity component in the SUT1 disruption strain grown on glucose. SUT2 and SUT3 had higher affinities for xylose and were expressed under aerobic conditions only. They had a substrate- concentration-modulated affinity for glucose, similar to that observed for HXT2 in S. cerevisiae (Reifenberger et al. 1997). The Michaelis constant for the low-affinity xylose transport in P. stipitis was different from that for SUT1 determined in an HXT1-7 deletion

strain in S. cerevisiae (Weierstall et al. 1999). This suggested that the low-affinity component could in fact be the superposition of several individual transporters. Therefore, it would be necessary to refine the kinetic constants for xylose transporters from various species once all the corresponding genes were cloned and expressed in a model system, such as the S. cerevisiae hxt1-7 strain.