Submitted to: Society of Industrial Microbiology Symposium
Publication Type: Abstract Only
Publication Acceptance Date: November 9, 2005
Publication Date: N/A
Technical Abstract: Trichoderma reesei (Hypocrea jecorina) is one of the most commonly used fungi for the manufacturing of enzyme products. These products are widely used in food, feed, pulp and paper, biomass conversion, and textile processes. Because of the long use in fermentation, the fungus has gained GRAS status. Some species of anaerobic fungi produce broad spectra of plant cell wall-degrading enzymes. Although the enzymes of anaerobic fungi are produced at concentrations (e.g., mgs per liter) too low to be applied directly to industrial applications, their specific activities are usually much higher than the counterparts of aerobic fungi. If these enzymes could be produced in large quantities, one might be able to demonstrate their utility in the above industries. Genes coding for the enzymes of anaerobic fungi are particularly A and T rich, but highly expressed genes of T. reesei are usually G and C rich. Initial attempts to express a xylanase gene (xyn11A) of Orpinomyces PC-2, a highly fibrolytic anaerobic fungus, in T. reesei were unsuccessful. Codon shifting the xyn11A gene allowed for expression and secretion of the enzyme by T. reesei using the cel7A promoter. The active fusion protein consisted of the codon shifted xyn11A gene fused to the cel5A sequence coding for the signal peptide, cellulose binding module, and linker. The size of the fusion protein was much higher than the calculated molecular mass, indicating that the heterologous xylanase is glycosylated. Engineering of a Kex2 protease recognition site between the linker and Xyn11A sequence failed to remove the fusion Cel5A polypeptide from the xylanase. Properties of the T. reesei and Escherichia coli produced forms of Xyn11A will be presented.