Submitted to: International Trichoderma Gliocladium Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 4/8/2006
Publication Date: 4/8/2006
Citation: Li, X., Jordan, D.B., Ximenes, E.A., Cotta, M.A., Dien, B.S., Hughes, S.R. 2006. Codon optimization of an A+T rich xylanase gene from the anaerobic fungus Orpinomyces PC-2 for expression by Trichoderma reesei [abstract]. In: Proceedings of the 9th International Workshop on Trichoderma and Gliocladium, April 6-8, 2006, Vienna, Austria. Paper No. T3.
Technical Abstract: Trichoderma reesei (Hypocrea jecorina) has been widely used for the manufacturing of industrial enzyme products because genetic modification and large-scale fermentation techniques for the fungus have been well developed. Anaerobic fungi, isolated from digestive tracts of herbivores, produce a broad spectrum of plant cell wall-degrading enzymes. Some of the cell wall-degrading enzymes exhibit high specific activity, but the levels of production by their natural hosts are low (e.g., mgs per liter); the genes are highly A+T rich, unlike the highly expressed genes in Trichoderma and Aspergillus. Possibly due to the rich A+T composition, initial attempts to express a xylanase gene (xynA) of Orpinomyces PC-2 in T. reesei were unsuccessful. After fusing the xynA with increased G+C composition to a region coding for the host Cel5A signal peptide, cellulose binding module, and linker, an active xylanase was secreted by T. reesei using the cel7A promoter. The active xylanase was detected only when the culture was maintained above pH 4.5; lower pH resulted in degradation of the recombinant xylanase. Engineering of a Kex2 protease recognition site between the linker and XynA sequence allowed the removal of the fusion Cel5A polypeptide and secretion of the xylanase. Purification and characterization of the T. reesei produced Orpinomyces XynA will also be presented.