Thesis (PhD)--University of Stellenbosch, 1999. / 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. / AFRIKAANSE OPSOMMING: Hemisellulose, wat hoofsaaklik uit xilaan bestaan, is ná sellulose, die volopste
hernubare polisakkaried in landbouafval. Xilaan is 'n komplekse polimeer wat
bestaan uit 'n β-D-1,4-gekoppelde xilopiranoseruggraat wat in sommige gevalle ook
sykettings bevat. Ensimatiese afbraak van xilaan benodig die werking van hele
aantal hidrolitiese ensieme.
Die gis Saccharomyces cerevisiae word al vir baie jare in die voedsel- en
drankbedryf (bak van brood en die maak van bier en wyn), asook vir die produksie
van etanol (vir menslik gebruik en as brandstof aanvuller) en enkelselproteïene
(proteïenaanvulling vir mens en dier) gebruik en het daarom volledige GRAS
(Generally Regarded As Safe) status. Ongelukkig kan S. cerevisiae nie komplekse
polisakkariede, xilaan ingesluit, afbreek of as koolstofbron benut nie. Met behulp
van rekombinante-DNA-tegnologie kan S. cerevisiae gekomplementeer word met
die nodige gene wat kodeer vir polisakkariedafbrekende ensieme om sodoende die
gis in staat te stel om 'n wyer verskeidenheid van substrate af te breek en te benut.
Dit sal lei tot die direkte bio-omskakeling van polisakkariede na bruikbare produkte
soos etanol, proteïenaanvullers en ensieme vir industriële gebruik.
In hierdie proefskrif word die suksesvolle uitdrukking asook die gesamentlike
uitdrukking van 'n xilanasegeen (Trichoderma reesei xyn2) en twee
β-xilosidasegene (Bacillus pumilus xynB en A. niger xlnD) in S. cerevisiae beskryf.
Multikopie episomale plasmiede pJC1, pDLG1, pDLG4 en pRLR1 met die glukose
onderdrukbare alkoholdehidrogenase 2 (ADH2) of die konstitutiewe
fosfogliseraatkinase 1 (PGK1)- promoter en -termineerder is vir hierdie doel
gebruik.
Verhoogde produksie van die rekombinante ensieme deur S. cerevisiae in 'n ryk
medium, sonder dat die gis die episomale plasmiedvektore verloor is moontlik
gemaak deur die urasielfosforibosieltransferasegeen (FUR1) van hierdie giste te
onderbreek met behulp van die LEU2-geen. Op hierdie manier word daar outomaties vir giste wat die URA3-uitdrukkingsplasmiede bevat geselekteer, selfs
in ryk medium.
Hoë vlak uitdrukking van T. reesei se xilanasegeen het S. cerevisiae in staat gestel
om xilaan tot kort xilo-oligosakkariede af te breek, maar byna geen monomeriese
D-xilose is gevorm nie. Albei die β-xilosidasegene het die gis in staat gestel om
kort xilo-oligosakkariede soos xilobiose en xilotriose na D-xilose af te breek. Die
gesamentlike uitdrukking van die xilanasegeen en B. pumilus se β-xilosidase geen
het 'n klein toename in die xilanase-aktiwiteit tot gevolg gehad, maar 'n drastiese
afname in die β-xilosidase-aktiwiteit. Hierdie rekombinante ras kon dus nie xilaan
tot xilose afbreek nie. Uitdrukking van T. reesei se β-xilanasegeen saam met die
β-xilosidasegeen van A. niger, het S. cerevisiae in staat gestel om xilaan tot sy
monomeriese boustene, D-xilose, af te breek.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/8490 |
| Date | 12 1900 |
| Creators | La Grange, Daniel Coenrad |
| Contributors | Van Zyl, W. H., University of Stellenbosch. Faculty of Science. Department of Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 225 p. : ill. |
| Rights | University of Stellenbosch |
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