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.
An NADH-dependent L-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora. The enzyme is part of the yeast pathway for L-arabinose catabolism. A fungal pathway for L-arabinose utilization has been described previously for molds. In this pathway L-arabinose is sequentially converted to L-arabinitol, L-xylulose, xylitol, and D-xylulose and enters the pentose phosphate pathway as D-xylulose 5-phosphate. In molds the reductions are NADPH-linked, and the oxidations are NAD ؉ -linked. Here we show that in A. monospora the pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by L-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. The ALX1 gene encoding the NADH-dependent Lxylulose reductase is strongly expressed during growth on L-arabinose as shown by Northern analysis. The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized. The reversible enzyme converts Lxylulose to xylitol. It also converts D-ribulose to Darabinitol but has no activity with L-arabinitol or adonitol, i.e. it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the L-configuration and the hydroxyl group of C-3 is in the D-configuration. It also has no activity with C-6 sugars or sugar alcohols. The K m values for L-xylulose and D-ribulose are 9.6 and 4.7 mM, respectively. To our knowledge this is the first report of an NADH-linked L-xylulose reductase.
The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.
Deletion of the phosphoglucose isomerase gene, PGI1, in Saccharomyces cerevisiae leads to a phenotype for which glucose is toxic. This is related to overproduction of NADPH through the oxidative part of the pentose phosphate pathway and the incompetence of S. cerevisiae to deal with this overproduction. A similar deletion (rag2) in Kluyveromyces lactis does not lead to such a phenotype. We transformed a genomic library of K. lactis in a yeast vector to a S. cerevisiae strain with a pgi1 deletion and screened for growth on glucose. We found a gene (GDP1) which encodes a phosphorylating glyceraldehyde-3-phosphate dehydrogenase, NADP-GAPDH (EC 1.2.1.13), that accepts both NADP and NAD. This is the first report of a eukaryotic, nonplant, NADP-linked GAPDH. Presumably, operation of this enzyme in the reverse direction enabled the transformed S. cerevisiae pgi1 deletion mutant to reoxidize the excess NADPH produced when glucose catabolism was forced through the pentose pathway. On the other hand, transcription of the gene in K. lactis was upregulated during growth on D-xylose, which suggests that in K. lactis the enzyme is involved in regeneration of NADPH needed for xylose assimilation, but transcription was not detected in a rag2 mutant grown on glucose. The presence of an asparagine (Asn46 in NADP-GAPDH) instead of the conserved aspartate found in related but NAD-specific enzymes may explain the ability of NADP-GAPDH to work with NADP as well as NAD.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.