Molecular cloning and functional expression of a novelNeurospora crassa xylose reductase inSaccharomyces cerevisiae in the development of a xylose fermenting strain

Abstract

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 NCAR-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.

Transcriptional comparison of the filamentous fungus Neurospora crassagrowing on three major monosaccharides D-glucose, D-xylose and L-arabinose

“…In fungi, D-xylose is converted to D-xylitol, then to D-xylulose and finally phosphorylated to D-xylulose-5-phosphate, which goes into the pentose phosphate pathway, via the enzymes D-xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XKS), respectively. The characteristics of XR and XDH from different species have been analyzed and significant improvement of xylose-fermentating yeast has been achieved by engineering of XR and XDH [2,3]. Many fungi can use D-xylose as a sole carbon source, and 14 xylose-fermenting fungal genomes have been sequenced and compared [4].…”

Developing elite Neurospora crassa strains for cellulosic ethanol production using fungal breeding

“…Various attempts have been made to reduce costs and labor of cellulosic ethanol production via CBP, however, these attempts have utilized organisms engineered to incorporate cellulolytic or fermentation pathways from other species [10, 14, 16, 17, 25]. While investigations aimed at understanding cellulolytic and fermentative systems, and even attempts at CBP have been made utilizing wild-type and gene deletion mutants of N. crassa , none have investigated natural variation due to allelic effects as a source of elite strains for CBP [8, 18, 27, 30, 31].…”

“…On the other hand, the main fungus used to generate the exoenzymes responsible for decomposition of cellulose to simple sugars, T. reesei , is ill-equipped to ferment the sugars it releases from cellulose through the process of saccharification [33]. In attempts to overcome these hurdles researchers have attempted to engineer organisms that are capable of robust cellulase expression and efficient fermentation of both hexose and pentose sugars to consolidate the production stages into one-stage CBP [5, 13, 16, 17, 25]. There are, however, filamentous fungi capable of producing the exoenzymes to decompose cellulosic plant biomass to simple sugars, along with the full sets of enzymes required to ferment both the hexose and pentose sugars of plant biomass to ethanol; among them is the model filamentous fungus Neurospora crassa [3, 7, 35, 36].…”

Development and characterisation of a recombinantSaccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties

“…The sources and relevant genotypes of bacterial and yeast strains, together with the plasmids used in this study, are listed in Table 1. Escherichia coli DH5α was used for the amplification of the yeast integrating plasmid pUSM 1001 (Thanvanthri Gururajan et al, 2007). This plasmid carries two gene cassettes, containing the P. stipitis xylose reductase (ADH1 PPsXYL1-ADH1 T ) and xylitol dehydrogenase (PGK1 P -PsXYL2-PGK1 T ) genes.…”