Browsing by Author "La Grange, Daniel Coenrad"
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- ItemGenetic engineering of the yeast Saccharomyces cerevisiae to degrade xylan(Stellenbosch : University of Stellenbosch, 1999-12) La Grange, Daniel Coenrad; Van Zyl, Willem Heber; University of Stellenbosch. Faculty of Science. Department of Microbiology.ENGLISH ABSTRACT: Hemicellulose, consisting mainly of xylan, ranks after cellulose, as the most abundant group of renewable polysaccharides in agricultural biomass. Xylan is a complex polymer consisting of a β D 1,4 linked xylopyranoside backbone, which may contain substituents. Enzymatic hydrolysis of xylan involves the action of a number of different hydrolytic enzymes. The yeast Saccharomyces cerevisiae has been used extensively in traditional food and beverage processes (baking, brewing and winemaking), as well as for the production of ethanol (potable alcohol and fuel extenders) and single-cell protein (protein supplements in food and animal feed). S. cerevisiae therefore has complete GRAS (Generally Regarded as Safe) status. However, the yeast S. cerevisiae can neither degrade nor utilize complex polysaccharides, including xylan. Through recombinant DNA technology, S. cerevisiae can be complemented by heterologous polysaccharase-encoding genes, thereby broadening its substrate range and facilitating a direct bioconversion of polysaccharides to valuable commodities, such as potable ethanol, protein supplements and industrial enzymes. In this study, the successful expression and co-expression of a β xylanase gene (Trichoderma reesei xyn2) and two β xylosidase genes (Bacillus pumilus xynB and A. niger xlnD) in S. cerevisiae, is described. Expression of these genes was obtained with the aid of multi-copy episomal yeast plasmids pJC1, pDLG1, pDLG4 and pRLR1. These plasmids contain either the derepressible alcohol dehydrogenase 2 (ADH2) or the constitutive phosphoglycerate kinase 1 (PGK1) promoter and terminator sequences. The enhanced production of recombinant enzymes by S. cerevisiae in a rich medium, without the risk of losing the episomal vector, was obtained by disrupting the uracil phosphoribosyltransferase (FUR1) gene in the plasmid-containing S. cerevisiae strains. This step ensured auto-selection of the URA3-bearing expression plasmids in rich growth medium. High level expression of the T. reesei β xylanase gene in S. cerevisiae enabled the yeast to degrade xylan to short xylo-oligosaccharides, but very little monomeric D xylose was formed. Both β xylosidase genes enabled S. cerevisiae to degrade short xylo-oligosaccharides like xylobiose and xylotriose. Co-expression of the β xylanase and the B. pumilus β xylosidase led to a small increase in the β xylanase activity, but a substantial decrease in the amount of β xylosidase activity. This recombinant yeast strain was unable to degrade xylan to D xylose. Expression of the T. reesei β xylanase with the A. niger β xylosidase gene enabled this strain to completely degrade xylan to its monomeric constituents, D xylose.