Recombinant bleaching enzymes from thermophiles expressed in fungal hosts

Bulk production of hemicellulases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases, and produces gram amounts of almost pure xylanase A from Dictyoglomus thermophilumin the medium in a chemostat culture. Both fungal and bacterial thermostable xylanases have been successfully expressed in T reesei. Efficient expression of the AT-rich xynB gene of Dictyoglomus thermophilum required reconstruction of the gene according to T reesei codon preferences. Unlike the situation found in K. lactis, a variety of post-translationally-modified species of XynB were visualized on gels. Removal of the N-linked glycosylation sites did not remove all of the iso-forms of XynB, suggesting that other post-translational modifications such as O-linked glycosylation also may be responsible for the multiple protein bands. In a protease-deficient strain of T. reesei, removal of all the N-linked glycosylation sites appeared to result in increased yields of XynB secreted into the culture medium. A glucose nonrepressible promoter has been isolated to complement the use of the cbh1 promoter, and combined with a fluorescent protein marker to aid in the identification of transformants and facilitate promoter comparison.