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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.

Production of ethanol from l-arabinose by Saccharomyces cerevisiae containing a fungal l-arabinose pathway

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.

Cloning of Two Genes (LAT1,2) Encoding Specific l-Arabinose Transporters of the l-Arabinose Fermenting Yeast Ambrosiozyma monospora

We identified and characterized two genes, LAT1 and LAT2, which encode specific l-arabinose transporters. The genes were identified in the l-arabinose fermenting yeast Ambrosiozyma monospora. The yeast Saccharomyces cerevisiae had only very low l-arabinose transport activity; however, when LAT1 or LAT2 was expressed, l-arabinose transport was facilitated. When the LAT1 or LAT2 were expressed in an S. cerevisiae mutant where the main hexose transporters were deleted, the l-arabinose transporters could not restore growth on d-glucose, d-fructose, d-mannose or d-galactose. This indicates that these sugars are not transported and suggests that the transporters are specific for l-arabinose.