Since the 1980s, modern molecular genetic techniques have been applied to a number of filamentous fungi.1,2 These methods have led to the development of various fungal hosts, such as various species of Aspergillus, Trichoderma, Neurospora, Fusarium, and Chrysosporium. With modern technologies, improved production of both native and non-native proteins can be achieved, shortening product development cycles and leading to greater exploitation of the physiological attributes that make fungi efficient protein producers.STRATEGIES FOR MAXIMIZING PROTEIN YIELDS
Molecular cloning for high-level expression and secretion. To express endogenous or heterologous genes, the promoter of the gene to be expressed is generally replaced by a promoter sequence from a highly expressed gene of the fungal host organism or a closely related organism. This replacement results in higher expression levels and the ability to work under well-established and optimized fermentation conditions. Constitutive promoters from the highly expressed genes of central metabolic pathways—such as glycolytic genes—are common. Alternatively, inducible promoters from highly expressed genes specific to the particular host are also used frequently. Examples include those from the Aspergillus niger glucoamylase encoding gene (glaA); the A. oryzae α-amylase encoding gene (amy); and the Trichoderma reesei or Chrysosporium lucknowense cellobiohydrolase I encoding genes (cbh1). For a new fungal host, these promoters can be validated by analyzing the expression levels of single-copy integrants of reporter gene constructs at a specific locus. Another validation option is identifying host-specific strong promoters by determining the major secreted proteins in the new fungal host grown under various culture conditions. The expression signals from those genes will often be useful for the expression of other genes, whether from the host organism itself or from different organisms.
Gene expression constructs must then be introduced into cells of the fungal host. In fungi there are three major gene-transfer strategies: treating fungal cells with lytic enzymes to create protoplasts; biolistic bombardment; and Agrobacterium-mediated transformation. Various transformation frequencies, some of them yielding up to several thousands transformants per microgram of DNA, have been reported.1