The first xylose-utilizing strains of S. cerevisiae were generated by expressing the Pichia stipitis genes XYL1 [23] and XYL2 [24], encoding XR and XDH, re­spectively [46-48]. P stipitis was chosen as the source of the heterologously expressed enzymes because it produces ethanol from xylose with theoret­ical yield, albeit only under well-controlled oxygen limitation [47,49,50], while most other naturally xylose-fermenting yeasts produce considerable amounts of the by-product xylitol [50]. Xylitol formation is a consequence of the inability of the cell to oxidize reduced cofactors in the absence of oxygen [32]. Contrary to XRs from most xylose-utilizing yeasts, XRs from P stipitis, Pachysolen tannophilus, and Candida shehatae can use not only NADPH but also NADH as a cofactor [21], which permits recirculation of the cofactors between the first two steps of the xylose pathway (Fig. 1).

Nevertheless, the first S. cerevisiae strains expressing the P stipitis XR and XDH produced xylitol, and the ethanol yield from xylose was low [47,48]. This was ascribed to the preference for NADPH over NADH of the XR [23]. Much research has been devoted to developing metabolic engineering strate­gies to improve xylose fermentation by XR — and XDH-carrying strains, often guided by the early suggestions to express either a strictly NADH-specific XR activity [32] or to express a transhydrogenase activity [21]. Both approaches are further discussed in the following sections together with other metabolic engineering strategies. Kinetic modeling estimated that the conversion of xylose to xylulose required a ratio of 1:10 of the initial XR and XDH activ­ities [51], which has been experimentally supported by several independent investigations [51-54]. The higher level of XDH is necessary to “pull” the xy­lose toward central metabolism [55], especially since the equilibrium of the XDH reaction favors xylitol formation [56]. In addition, it has more recently been found that efficient xylose metabolism requires high activity of both XR and XDH [54,57].

3

Arabinose

3.1