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 CO2 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 CO2 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 CO2 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 CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.

FIG. 1.

Redox cofactors in the metabolic pathway for d-xylose fermentation. d-Xylose is converted to d-xylulose through an NADPH-utilizing reductase and NAD⁺-utilizing dehydrogenase. d-Xylulose is then phosphorylated to the pentose phosphate intermediate d-xylulose 5-phosphate (X5P). The products of the pentose phosphate pathway are fructose-6-phosphate (F6P) and GAP. GAP is reduced through an NADP-GAPDH, encoded by GDP1, or by the endogenous NAD-GAPDH, depending on cofactor availability. In the following reactions involving an NADH requiring alcohol dehydrogenase, equimolar amounts of CO₂ and ethanol are derived. A competing pathway for NADP⁺ is the oxidative part of the pentose phosphate pathway. Glucose-6-phosphate (G6P) is derived from fructose-6-phosphate and can enter the oxidative part of the pentose phosphate pathway through G6PDH, which is encoded by the ZWF1 gene. G6P is oxidized, thereby generating NADPH and CO₂. The deletion of ZWF1 prevents this reaction.

FIG. 2.

Ethanol production in the fermenter cultivations. Pure d-xylose is fermented under anaerobic conditions. The ethanol production is normalized to the biomass. The average biomass is indicated in Table 3. DM, dry mass.

FIG. 3.

Xylitol production in the fermenter cultivations. Conditions are as described for Fig. 2 and Table 3. DM, dry mass.