Engineering of Pichia stipitis for enhanced xylan utilization

[摘要] ENGLISH ABSTRACT:Plant biomass, the most abundant renewable resource in nature, consists of matrices ofmainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, themajor hemicellulose component in plant cell walls, is the most abundant polysaccharideafter cellulose. This makes the main constituent sugar of xylan, D-xylose, the secondmost abundant renewable monosaccharide in nature. Very few hemicelluloses are eitherhomopolymeric or entirely linear. Therefore, the variety of enzymes involved in theirhydrolysis is more complex than the enzyme group responsible for the hydrolysis ofcellulose. Although the ability to degrade xylan is common among bacteria andfilamentous fungi, this trait is relatively rare among yeasts. However, some strains of theyeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also oneof the best D-xylose fermenting yeasts thus far described, this yeast has the potential offermenting polymeric xylan directly to ethanol. However, it was shown that the naturalxylanolytic ability of this yeast is very weak.In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeastto produce heterologous proteins, and to determine the enhancement of xylan utilisationby the recombinant strain. The native xylose reductase gene (XYLl) and transketolasegene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase(PGKl) gene promoter were cloned into P. stipitis transformation vectors and used toexpress the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It wasshown that the XYLl promoter was induced in the presence of D-xylose and that the TKLpromoter was constitutively expressed. The PGKl promoter of S. cerevisiae did notfunction 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 ~-xylanaseactivity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activitydeclined 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 alleviatethe reduction of heterologous l3-xylanase activity. When the Aspergillus nigerl3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02)with the S. cerevisiae mating factor secretion signal (MFal) under control of theP. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml wasobserved. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidaseactivities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC andXL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124nkatlml, respectively.The expression of the fungal xylanolytic genes in P. stipitis also led to an increasedbiomass yield when the recombinant strains were cultured on birchwood xylan as solecarbon source. The strain co-expressing the A. kawachii l3-xylanase and A. nigerl3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomasslevel than the control strain. Biomass levels of the recombinant strains were furtherimproved on average by 85% by growing them in a fermentor under conditions of highoxygenation. The strains were also tested for direct conversion of xylan to ethanol andthe strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encodinggenes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yieldover the reference strain. These strains represent a step towards the efficient degradationand utilisation of hemicellulosic materials by ethanol-producing yeasts.

[发布机构] Stellenbosch University