Engineering Redox Cofactor Regeneration for Improved Pentose Fermentation in Saccharomyces cerevisiae

Abstract

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses d -xylose and l -arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD + , which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase ( ZWF1 ). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1 , which codes for a fungal NADP + -dependent d -glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the d -xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO 2 production. The resulting strain fermented d -xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1 ; i.e., the levels of the unwanted side products xylitol and CO 2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO 2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD + . The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated d -xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO 2 from d -xylose to a strain that produced mainly ethanol under anaerobic conditions.