The problem of cofactor regeneration has also been addressed by engineering reactions distant from the xylose utilization pathway, as demonstrated by dif­ferent approaches to introduce a transhydrogenase function in S. cerevisiae. Heterologous expression of a bacterial transhydrogenase [118] in S. cerevisiae carrying XR and XDH reduced xylitol formation, but also increased glycerol, rather than ethanol formation [116] (strain TMB3254, Table 2), indicating that the transhydrogenase reaction did not proceed in the direction favorable for ethanolic xylose fermentation [116,118].

Intracellular cofactor concentrations have also been altered, introduc­ing in S. cerevisiae the NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis [119] (strain H2673, Table 1). When the ZWF1 gene was simultaneously deleted, the expression of GAPDH improved ethanol formation [115] (strain H2684, Table 1). Similarly, when a NAD(P)+-dependent nonphosphorylating GAPDH from Streptococ­cus mutans was overexpressed in an XR-XDH-XK-carrying strain, increased ethanol formation was observed [120] (strain CPBCB4, Table 3). The result suggested that less carbon was lost as carbon dioxide when NADPH was formed outside the oxidative PPP and that NAD+ consumption in the lower glycolysis was simultaneously reduced.

Engineering the ammonium assimilation pathway [121] has also been used to modify the intracellular cofactor concentrations. Based on the as­sumption that NADH would be used for ammonium assimilation to generate NAD+ for the XDH reaction, the NADPH-dependent glutamate dehydroge­nase gene GDH1 was deleted, and an NADH-dependent isoenzyme (GDH2) was overexpressed. Reduced xylitol formation and higher ethanol forma­tion were observed [121] (strain CPB. CR4, Tables 1 and 3). Alternatively, the GS-GOGAT complex coded by the genes GLT1 and GLN1 was overex­pressed, which only affected xylose fermentation in a continuous fermenta­tion setup [121] (strain CPB. CR5, Tables 1 and 3).

4.6