Engineering of Pichia stipitis for enhanced xylan utilization

Den Haan, Riaan (2003-12)

Thesis (PhD)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the major hemicellulose component in plant cell walls, is the most abundant polysaccharide after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second most abundant renewable monosaccharide in nature. Very few hemicelluloses are either homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their hydrolysis is more complex than the enzyme group responsible for the hydrolysis of cellulose. Although the ability to degrade xylan is common among bacteria and filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of fermenting polymeric xylan directly to ethanol. However, it was shown that the natural xylanolytic ability of this yeast is very weak. In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast to produce heterologous proteins, and to determine the enhancement of xylan utilisation by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase (PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not function in P. stipitis . When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene (xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity declined over time due to the presence of extracellular proteases, secreted by P. stipitis. Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02) with the S. cerevisiae mating factor secretion signal (MFal) under control of the P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124 nkatlml, respectively. The expression of the fungal xylanolytic genes in P. stipitis also led to an increased biomass yield when the recombinant strains were cultured on birchwood xylan as sole carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass level than the control strain. Biomass levels of the recombinant strains were further improved on average by 85% by growing them in a fermentor under conditions of high oxygenation. The strains were also tested for direct conversion of xylan to ethanol and the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield over the reference strain. These strains represent a step towards the efficient degradation and utilisation of hemicellulosic materials by ethanol-producing yeasts.

AFRIKAANSE OPSOMMING: Plant biomassa, die volopste hernubare koolstotbron in die natuur, bestaan uit matrikse van lignien, sellulose en hemisellulose. Xilaan, die hoof hemisellulose komponent in plantselwande, is na sellulose die volopste polisakkaried. Gevolglik is die hoof suikerkomponent van xilaan, naamlik D-xilose, die tweede volopste hernubare monosakkaried in die natuur. Baie min hemisellulose molekules is homopolimere of heeltemal linieêr. Daarom is die ensieme betrokke by die atbraak van hemiselluloses meer kompleks as die ensieme betrokke by die atbraak van sellulose. Bakterieë en filamentagtige fungi wat oor die vermoë om xilaan af te breek beskik, kom wydversprei voor maar relatief min giste kan xilaan benut. Sommige rasse van die gisspesie Pichia stipitis het egter beperkte vermoë om xilaan af te breek. P. stipitis is ook een van die beste D-xilose fermenterende giste wat tot dusver beskryf is en het dus die potensiaalom etanol vanafpolimeriese xilaan te produseer. In hierdie studie is gene wat kodeer vir xilaanatbrekende ensieme in P. stipitis uitgedruk om die vermoë van die gis as heteroloë uitdrukking sisteem te evalueer. Verder is die effek van die heteroloë xilaanatbrekende ensieme tydens groei op xilaan as enigste koolstotbron getoets. Die promoters van die xilosereduktasegeen (XYLl), die transketolasegeen (TKL) van P. stipitis en die fosfogliseraatkinasegeen (PGKl) van Saccharomyces cerevisiae is in P. stipitis transformasie vektore gekloneer en gebruik om die Trichoderma reesei ~-xilanasegeen (xyn2) as verklikkergeen uit te druk. Dit het bewys dat die XYLI promotor induseerbaar is in die teenwoordigheid van D-xilose terwyl die TKL geen konstant uitgedruk was. Die PGKI promotor van S. cerevisiae was nie funksioneel in P. stipitis nie. Ekstrasellulêre ~-xilanase aktiwiteit van onderskeidelik 136 nkatlml en 171 nkatlml kon waargeneem word wanneer die T reesei xyn2 geen of die Aspergillus kawachii ~-xilanasegeen (xynC) onder beheer van die XYLI promotor uitgedruk is. Hierdie aktiwiteit het afgeneem na gelang van tyd a.g.v. die teenwoordigheid van ekstrasellulêre proteases wat deur P. stipitis uitgeskei word. Die afname van ekstrasellulêre ~-xilanase aktiwiteit kon nie voorkom word deur die kulture in 'n fermentor te groei en die pH vlak tot pH 6 te beheer nie. Tydens uitdrukking van die Aspergillus niger ~-xilosidase geen (xlnD) as 'n fusiegeen (genoem XL02) met die paringsfaktor sekresiesein (MFal) van S. cerevisiae onder transkripsionele beheer van die P. stipitis TKL promotor, kon ekstrasellulêre ~-xilosidase aktiwiteit van 0.132 nkatlml waargeneem word. Gesamentlike uitdrukking van die xyn2 en XL02 gene het gelei tot ~-xilanase en ~-xilosidase aktiwiteite van 128 nkatlml and 0.113 nkat/ml, onderskeidelik. Gesamentlike uitdrukking van die xynC en XL02 gene het gelei tot ~-xilanase en ~-xilosidase aktiwiteite van 165 nkatlml and 0.124 nkatlml, onderskeidelik. Die uitdrukking van xilaanatbrekende ensieme III P. stipitis het verhoogbe biomassaproduksie teweeg gebring wanneer die rekombinante gisrasse op birchwood xilaan as enigste koolstotbron gegroei het. Die rekombinante ras wat die A. kawachii ~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitdruk, was die mees suksesvolle ras en het 3.2-voudig hoër biomassa as die kontrole ras opgelewer. Die biomassa van die rekombinante rasse tydens groei op xilaan as enigste koolstotbron kon gemiddeld met 85% verhoog word deur die giste onder hoë suurstotkonsentrase in 'n fermentor te kweek. Die rekombinante rasse is verder ook getoets vir hul vermoë om xilaan direk tot etanol om te skakel. Die rekombinante ras wat die A. kawachii ~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitgedruk het, het 'n 3.6- voudige verhoging in etanolproduksie getoon en 1.35 gIL ethanol gelewer. Hierdie rekombinante gisrasse verteenwoordig 'n stap nader aan die doeltreffende atbraak en benutting van hemisellulose deur etanolproduserende giste.

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