5-05: Industrial level production of enzymes by Trichoderma reesei for cellulosic bioethanol

Efficient expression is the key for economically-viable bulk production of enzymes. Filamentous fungi naturally secrete large amounts of proteins into the growth medium and are commonly used for the large-scale manufacture of proteins, particularly industrial enzymes (1). However, although other fungal proteins are efficiently expressed, expression of gene products from other organisms such as thermophilic bacteria, is subject to a number of bottlenecks that reduce yield. Following a proteomic analysis of the Trichoderma secretome, we constructed four expression vectors that utilise either the cbh2 or egl2 promoters. Thermophilic xylanases are of particular interest for efficient hydrolysis of the hemicellulosic component of woody materials into fermentable pentose sugars (2). The xynB gene encoding xylanase B from the thermophilic bacterium Dictyoglomus thermophilum has been inserted into the vectors for heterologous expression as a model system. This enzyme is particularly effective in the hydrolysis of both soluble and insoluble xylan in hemicellulose. The codon usage of the xynB gene has been modified for expression in T. reesei. Expression of xynB from the pEG2-cbmlin and pCBH2-sigpro vectors was found to be greater than that using the EG2-sigpro and CBH2-cbmlin constructions, based on zymogram analysis and liquid enzyme activity assays. We will discuss the ability of the new promoter constructions to drive xylanase production as influenced by the structural differences in the expression cassette motifs and their contribution to improved yields under fermentor conditions (1) Nevalainen et al (2005), Trends Biotechnol. 23: 468 (2) Viikari et al (2007), Adv. Biochem. Eng. Biotech. 108: 121