Molecular cloning and functional expression of a novel Neurospora crassa xylose reductase in Saccharomyces cerevisiae in the development of a xylose fermenting strain

The development of a xylose-fermenting Saccharomyces cerevisiae yeast would be of great benefit to the bioethanol industry. The conversion of xylose to ethanol involves a cascade of enzymatic reactions and processes. Xylose (aldose) reductases catalyse the conversion of xylose to xylitol. The aim of this study was to clone, characterise and express a cDNA copy of a novel aldose reductase {NCAR-X) from the filamentous fungus Neurospora crassa in S. cerevisiae. NCAR-X harbours an open reading frame (ORF) of 900 nucleotides. This ORF encodes a protein (NCAR-X, assigned NCBI protein accession ID: XP_956921) consisting of 300 amino acids, with a predicted molecular weight of 34 kDa. The NVAR-X-encoded aldose reductase showed significant homology to the xylose reductases of Candida tenuis and Pichia stipitis. When NCAR-X was expressed under the control of phosphoglycerate kinase I gene (PGK1) regulatory sequences in S. cerevisiae, its expression resulted In the production of biologically active xylose reductase. Small-scale oxygen-limited xylose fermentation with the NCAR-X containing S. cerevisiae strains resulted In the production of less xylitol and at least 15% more ethanol than the strains transformed with the P. stipitis xylose reductase gene (PsXYL1). The NCAR-X-encoded enzyme produced by S. cerevisiae was NADPH-dependent and no activity was observed in the presence of NADH. The co-expression of the NCAR-X and PsXYL1 gene constructs in S. cerevisiae constituted an important part of an extensive research program aimed at the development of xylolytic yeast strains capable of producing ethanol from plant biomass.