Various approaches to improve transcription have been used in fungi. For the expression of heterologous genes, especially
of nonfungal origin, codon-optimized, synthetic genes can improve the transcription rate. The gene is adapted to the preferred
codon usage of the host strain, based on the codon usage data of a large number of well-expressed or secreted proteins. To
obtain high- level expression of a particular gene, a well-established procedure is targeting multiple copies of the recombinant
gene constructs to the locus of a highly expressed endogenous gene. An advantage of such a gene replacement is knowing that
the integration occurs in a region of the genome that is actively transcribed. When the target locus is that of a highly-expressed
secreted protein, the reduced load on the secretion pathway may facilitate expression and secretion of the recombinant gene
For the secretion of foreign proteins, fusion strategies are used to facilitate translocation in the secretion pathway and
to protect the heterologous protein from degrading. An amino terminal fusion with an efficiently secreted protein is generally
preferred. A truncated form of the A. niger glucoamylase (GII) gene has been used successfully in several fungal hosts.3 To separate the fusion partner from the target gene, one can use the endogenous secretion machinery of the fungal host or
engineer cleavage sites between the fusion partner and target. During normal fungal protein secretion, passage of the protein
through the cell membrane is accompanied by proteolytic cleavage of the signal sequence from the mature protein in a sequence-specific
manner. When such a recognition sequence is engineered between the fusion partner and target protein, cleavage results, with
the secretion of the protein of interest separated from the fusion partner. Sometimes, however, it is desirable to maintain
intact fusion proteins, if they are more stable than the corresponding independent target protein. In those cases, protease
recognition sites that are not recognized by the cellular secretion machinery can be engineered between the secretion partner
and target protein, with subsequent in vitro cleavage and separation of the target from the fusion partner.
Reducing proteolytic degradation.
One of the major challenges in fungal biotechnology, especially for the production of pharmaceutical proteins, is that the
recombinant protein is often degraded by proteases of the fungal host. Reducing protease activities can be achieved by combining
different approaches. One involves selecting or screening for fungal host strains with low protease activity. This can be
accomplished either by direct selection, using "suicide" selection substrates, or by mutagenesis and screening for reduced
protease activity on indicator plates. Other approaches are identifying and inactivating genes for specific proteases, identifying
and modifying wide-domain protease regulators, and optimizing medium composition and fermentation protocols.4 The isolation of mutant strains that can produce higher levels of a specific heterologous protein with subsequent removal
of the expression construct is another strategy used to produce host strains with reduced proteolytic activity.5
Protein glycosylation is an important consideration in the production of eukaryotic proteins and is especially important
for therapeutics. Non-native glycosylation can influence serum half-life, function, and immunogenicity of therapeutic proteins.
Considerable progress has been made in modifying the glycosylation patterns of fungi, especially in the methylotrophic yeast
While there has been considerable effort in understanding glycan biosynthesis,3 the progress toward glycan modification in filamentous fungi is not as advanced as in Pichia. Modification of native fungal glycan structures to make them human-identical or human-compatible, whether by in vivo methods or by in vitro remodeling7 will be important if filamentous fungi are going to be viable alternative systems for the production of proteins of pharmaceutical
Figure 1. At left is a microscopic image of wild type Chrysosporium lucknowense. The filamentous nature of this organism results
in highly viscous cultures, surface matting, and clumping. A nonviscous mutant "propagule" is shown at right. The nonfilamentous
strain offers advantages for high-throughput screening, gene expression, and protein production.