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.

Genomics-driven Reconstruction of Acinetobacter NAD Metabolism

“…Moreover, understanding of NAD metabolism in A. baylyi is of additional importance for its biotechnological applications in biodegradation and manufacturing of biopolymers (46). Indeed, maintenance and regeneration of the NAD cofactor pool is a known bottleneck in many microbial fermentations (47,48).…”

Fermentation performance of engineered and evolved xylose‐fermenting Saccharomyces cerevisiae strains

“…Nevertheless, the principal stoichiometric redox problem was not solved (Bruinenberg et al, 1984;Hahn-Hägerdal et al, 2001), and thus xylitol formation will likely remain an inherent property of strains engineered with xylose reductase and xylitol dehydrogenase. Instead, conceptually new metabolic engineering strategies will be necessary such as the use of xylose isomerase (Kuyper et al, 2003;Walfridsson et al, 1996) or new biochemical routes to NADH reoxidation (Roca et al, 2003;Verho et al, 2003). Methods of mutagenesis and selection or more elaborate evolutionary engineering (Sauer, 2001) appear to be the most promising for initial introduction of a novel (Sonderegger and Sauer, 2003) or for improvement of a weak property (Steiner and Sauer, 2003).…”

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

“…3) and furfural in the fermentation broth (data not shown). The increase in xylitol production was likely due to predominantly xylose being consumed and a co-factor imbalance in the XR/ XDH pathway [19].…”

“…This has been attributed to capacity limitations in the pentose phosphate shunt [9] and mismatched co-factor dependency during xylose catabolism in engineered XR/XDHstrains [17]. The co-factor imbalance between NAD(P)H-consuming XR and NADH-producing XDH reactions effects the production of xylitol [18,19].…”