ABSTRACT
Filamentous fungi, particularly those of the genera Aspergillus and Trichoderma, are widely used for the heterologous expression of proteins as they are capable of producing up to 20-30 grams of protein per litre of culture (Kalsner et al. 1995, Archer and Peberdy 1997, Gouka et al. 1997, Withers et al. 1998, Maras et al. 1999). Filamentous fungi possess many other signifi cant advantages as protein expression systems, including ease of genetic manipulation, a high level of adaptability allowing exploitation of a wide range of environmental conditions and nutrient sources, well developed and diverse promoter systems and a structural morphology amenable to high cell culture density and simple biomass fi ltration (Lubertozzi and Keasling 2009). An important consideration in the production of non-fungal enzymes in fungal hosts is the fi delity of post-translational processing events, such as protein glycosylation (reviewed in Hamilton and Gerngross 2007, De Pourcq et al. 2010). The post-translational addition of non-authentic N-glycans to proteins used as therapeutic agents can result in reduced activity or stability, increased serum clearance and can sometimes result in an adverse immune response (Jenkins et al. 1996, Jenkins 2007, Jacobs and Callewaert 2009). To utilize fi lamentous fungi for the production of such specialized glycoproteins, it is preferable to produce glycoproteins which carry carbohydrate structures as similar to the natural product as possible. Manipulation of the N-glycosylation pathway to produce glycoproteins with ‘correct’ N-glycan structures would dramatically increase the utility of these expression systems for the production of therapeutic protein drugs. Engineering of glycan specifi c protein expression has been accomplished in several diverse systems, including: the yeasts Pichia pastoris (Choi et al. 2003, Bobrowicz et al. 2004, Vervecken et al. 2004, Wildt and Gerngross 2005, Hamilton and Gerngross 2007), Ogataea minuta, (Kuroda et al. 2006), Saccharomyces cerevisiae (Chiba and Jigami 2007, Chiba and Akeboshi 2009), Hansenula polymorpha (Oh et al. 2008) and Yarrowia lipolytica (Song et al. 2007); the fi lamentous fungi Trichoderma reesei (Maras et al. 1997b, Maras et al. 1999), Aspergillus spp. (Kainz et al. 2008); insect cell lines (Hollister
et al. 2002, Betenbaugh et al. 2004, Tomiya et al. 2004, Viswanathan et al. 2005, Shi and Jarvis 2007); plant cells (Bakker et al. 2001); and mammalian cell lines (Grabenhorst et al. 1999). Key to all of these expression systems is the correct production of both the type and complexity of N-glycans added to glycoproteins.
