In contrast to S. cerevisiae, many yeast species are capable of utilising xy­lose as the sole carbon and energy source for respiratory growth. However, only few of these yeasts are capable of fermenting xylose to ethanol under oxygen-limited conditions, such as for instance Pichia stipitis and Pachysolen tannophilus [65].

Maybe not surprisingly, xylose-metabolising yeasts have predominantly been isolated from wood-related environments. The pathway for D-xylose metabolism used by these yeasts to convert D-xylose to D-xylulose was first described in 1955 [25] and involves a two-step conversion that involves two oxidoreductases (Fig. 1): xylose reductase (EC 1.1.1.21) and xylitol dehydro­genase (EC 1.1.1.9). The xylose reductase has a strong preference for NADPH, whereas the subsequent oxidation of xylitol via xylitol dehydrogenase pro­duces NADH (Table 1).

Clearly, this difference in cofactor specificity can result in redox imbalance. To generate the NADPH for the xylose reductase reaction, part of the D-xylose carbon must be directed through the oxidative pentose phosphate pathway (involving the glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase reactions). While this results in a loss of some carbon as CO2,

which goes at the expense of the ethanol yield on D-xylose, it enables the efficient regeneration ofNADPH [16,32,45,69].

However, the cells have to take additional measures to reoxidise the ex­cess NADH generated in the xylitol dehydrogenase reaction. In the presence of oxygen, this excess NADH can be reoxidised by respiration. This will re­quire accurate dosage of oxygen to prevent full respiration of D-xylose. Such accurate control is difficult to envisage in large-scale processes for ethanol production, which should preferably involve a minimum of aeration to reduce costs.

Under anaerobic conditions, reoxidation of excess NADH can be ac­complished via the production of compounds that are more reduced than D-xylose, such as xylitol and/or glycerol. The production of xylitol occurs via xylose reductases, which have a dual co-enzyme specificity and thereby can also use NADH, or alternatively via other aspecific reductases. As this mech­anism involves the consumption of one D-xylose for each NADH generated, it has a tremendously negative impact on the ethanol yield from D-xylose [45]. Glycerol production is a well-known redox sink during hexose fermenta­tion and especially under anaerobic conditions, but requires both carbon and ATP [67].

The preference of xylose reductase for NADPH is not only species — but also strain-dependent (Table 1). The in vivo ratio ofNADPH over NADH utilisa­tion by xylose reductase and the redox balance requirements determine the

requirement for NADH sinks such as xylitol and glycerol (Fig. 2) in anaerobic cultures [14,69]. When this NADPH/NADH ratio equals zero, xylose reduc­tase only uses NADH and thereby consumes all NADH produced in the xylitol dehydrogenase reaction. Since in addition no regeneration of NADPH is re­quired for the xylose reductase reaction, redox-balanced xylose metabolism will occur according to Eq. 1:

Ratio = 0: 6 xylose ^ 10 ethanol + 10 CO2 + 10 ATP. (1)

At a ratio of one (Eq. 2), one out of every two D-xylose molecules can be further metabolised to ethanol, whereas the other is reduced to xylitol to maintain NADH balance. In addition, some carbon has to be redirected for the generation of NADPH, resulting in the formation of only 9 mol of ethanol from 12 mol of D-xylose (45% of the theoretical yield). Following these redox-balance considerations, catabolism via a xylose reductase with a NADPH/NADH-utilisation ratio of one will follow:

Ratio = 1: 12 xylose ^ 9 ethanol + 12 CO2 + 9 ATP + 6 xylitol. (2)

At ratios above one, NADH-dependent xylitol formation cannot compensate for the production of NADH in the xylitol dehydrogenase reaction and glycerol formation becomes essential for redox balancing [32,45,69]. When the xylose

reductase solely uses NADPH (an infinite NADPH/NADH ratio) this would result in the formation of only 0.5 mol ethanol per mol of xylose fermented.

Ratio = to : 6 xylose + 3 ATP ^ 3 ethanol + 6 glycerol + 6 CO2 . (3)

Despite these inherent redox restrictions and ensuing loss of ethanol yield on D-xylose, the expression of xylose reductase and xylitol dehydrogenase has long been the most successful strategy to enable D-xylose consumption by S. cerevisiae (elsewhere in this volume, and [29,32,33,39,63]). Although attempts have been made to change the cofactor specificity of xylose reduc­tase, fermentation properties of a S. cerevisiae strain containing this gene are not available [55]. Similarly, expression of a transhydrogenase in S. cere­visiae, with the aim of converting excess NADH into NADPH, did not result in reduced byproduct formation [51]. The latter result is perhaps not alto­gether surprising as, with NADPH/NADP+ ratios generally being higher than NADH/NAD+ ratios [51], reduction of NADP+ with NADH is thermodynam­ically unfavourable.

Despite the inherent redox constraints of S. cerevisiae strains based on the xylose reductase/xylitol dehydrogenase strategy, this strategy has resulted in many important insights into the kinetics of D-xylose metabolism by en­gineered S. cerevisiae strains. These findings include the benefits of over­expression of xylulokinase [29, 56], the side role of the S. cerevisiae aldose reductase (Gre3) (besides the heterologous dual specificity xylose reduc­tases) in xylitol formation [66], the role of the enzymes of the non-oxidative part of the pentose phosphate pathway [34,43], characterisation of D-xylose transport [27,62] and many studies on the inhibitor tolerance/sensitivity of D-xylose-consuming strains [54]. The latter will be especially crucial for suc­cessful application of D-xylose-consuming S. cerevisiae strains for ethanol production from lignocellulosic hydrolysates (see Sect. 7).

1.3