In contrast to the energy-conserving function of glycolysis, the main metabolic function of the PPP is to provide anabolic intermediates such as ribulose 5-phosphate, erythrose 4-phosphate, and NADPH for biosynthesis and cell growth. The flux through the nonoxidative PPP in S. cerevisiae was found to be much lower than in other yeasts [105], which was later con­firmed by metabolome analysis [106]. The low PPP activity in S. cerevisiae is sometimes interpreted to be a result of the domestication of S. cerevisiae by prolonged selection for carbon dioxide and ethanol production from hexose sugars. However, PPP activity is a crucial part of pentose metabolism, since it is virtually the only way to introduce xylulose into the central metabolism. It was early pointed out that the PPP activity may limit xylose metabolism in S. cerevisiae [47,107], which was further supported when excretion of PPP intermediates was observed in xylulose — and xylose-metabolizing S. cere­visiae [43,108].

The insufficient flux through the nonoxidative PPP in S. cerevisiae has been indirectly confirmed in several genome-scale and enzymatic analyses of mutant strains with improved xylose metabolism, where invariably ei­ther the transaldolase (TAL1) or the transketolase (TKL1) genes, or both, have been found to be upregulated [57,71,91,109-111]. Directly, the im­portance of the flux through the PPP has been confirmed by the superior pentose utilization and ethanolic fermentation by strains in which the en­zymes of the nonoxidative PPP have been overexpressed. An early attempt to overexpress P. stipitis transketolase in xylose-metabolizing S. cerevisiae was not successful [112], whereas overexpression of the endogenous S. cere­visiae transaldolase (TAL1) resulted in improved growth on xylose [78]. Later, the overexpression of all four nonoxidative PPP genes, including not only TAL1 and TKL1 but also ribulose-5-phosphate 4-epimerase (RPE1) and ribu — lokinase (RKI1), was shown to improve xylulose consumption by S. cere­visiae [90,113] (strain TMB3026, Table 3). Moreover, the improvement result­ing from the overexpression of the four genes was higher than when each gene was overexpressed alone [90]. The simultaneous overexpression of the whole nonoxidative PPP, together with GRE3 deletion, allowed growth on xylose in a strain carrying a bacterial XI [42] (strain TMB3050, Table 2). The usefulness of this combination of modifications was confirmed when it allowed aerobic and anaerobic growth on xylose in a strain carrying the Piromyces XI [97] (strain RWB217, Table 1). PPP overexpression also al­lowed superior xylose fermentation rates in combination with high levels of XR and XDH [42,54] (cf. strains TMB3057, TMB3056, and TMB3062, Table 1 and Fig. 5).

4.5