Richard P. Burlingame, PhD
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Filamentous fungi are widely used in industry to produce small-molecule metabolites. Examples include antibiotics like penicillin,
cholesterol-lowering drugs like Lovastatin, and food ingredients like citric acid. Because of their biological niche as microbial
scavengers, filamentous fungi are also uniquely adapted to produce and secrete proteins. In environments rich in biological
polymers, such as forest floors, the fungi thrive by secreting enzymes that degrade the polymers to produce monomers that
can be readily used as nutrients for growth. The natural ability of fungi to produce proteins has been widely exploited, particularly
in the production of industrial enzymes. While the levels of protein production in natural isolates are generally not high
enough for commercial exploitation, improved strains and processes can lead to enormous yield increases, making it possible
to produce yields of tens of grams of protein per liter of fermentation culture.
Jan C. Verdoes, PhD
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Traditionally, these yield increases have been achieved through mutagenesis and screening for increased production of proteins
of interest. While effective, this approach is useful only for the overproduction of endogenous proteins in isolates containing
the enzymes of interest. For each new protein product, a lengthy strain and process development program is required, often
with an organism for which there is little if any biochemical, physiological, or genetic knowledge.
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
