The observation that most xylose-utilizing fungi produce considerable amounts of xylitol from xylose, and that only species containing also the

NADH-dependent XR activity are capable of producing ethanol from it, sug­gested that the different cofactor preferences of XR and XDH limit ethanolic xylose fermentation by yeast [21,32]. Since S. cerevisiae ferments xylu­lose [1,2], it was suggested that xylose fermentation could be easily achieved by heterologous expression of an XI [32,33]. Indeed, xylose was fermented to ethanol when extracellular XI was added to the medium [33]. This enzyme, with activity not only for xylose but also for glucose, is industrially used for the production of high-fructose corn syrup (HFCS) [18] to convert starch — derived glucose into the sweeter sugar fructose to reduce the sugar demand in the food industry. Heterologous expression of bacterial XI genes in S. cere­visiae proved to be challenging, and for many years no actively expressed enzyme was reported [34-39]. The first functionally expressed XI in S. cere­visiae [40] originated from the bacterium Thermus thermophilus [41]. It was later shown that the low activity of the bacterial XIs in yeast could be par­tially related to intracellular precipitation [39], and it was suggested that the rigid nature of the thermotolerant T. thermophilus XI aided correct folding of the protein in S. cerevisiae. However, the activity of this enzyme at 30 °C was too low to allow xylose fermentation. Still, when combined with other genetic modifications, aerobic growth on xylose was demonstrated by S. cerevisiae carrying the T. thermophilus XI [42] (strain TMB3050, Table 2).

More recently, an XI from the obligate anaerobe rumen fungus Piro — myces [20] was expressed in S. cerevisiae with an activity of about 1 U/mg protein at 30 °C [43] (strain RWB202, Tables 3 and 4). Later, bacterial XIs with high sequence similarity to the Piromyces XI, such as those from Bac- teroides thetaiotaomicron [44] and Xanthomonas campestris [45], were also expressed in S. cerevisiae, but the activity of these enzymes in S. cerevisiae was lower than that of the Piromyces XI. Despite the relatively high activity of Piromyces XI in S. cerevisiae, the expression of this enzyme alone did only al­low very slow growth on xylose [43], suggesting that the conversion of xylose to xylulose does not alone control the xylose metabolism in S. cerevisiae [42]. This observation may also set in a new light the failures of early trials for het­erologous XI expression where, in many cases, functional XI expression was only assayed as growth on xylose [35,37].

2.3