18.10 Fungi as cell factories producing heterologous proteins
We have been using fungi to produce materials of commercial value for a long time (see Chapter 17) and there has always been a drive to improve the fungal strains. There are several strategies for this:
- Mutagenesis; meaning the use of chemical mutagens, ultraviolet light or radiation to generate in one step strains with improved expression and secretion of the product of interest. This has been used successfully to improve productivity of:
- α-amylase by Aspergillus oryzae (see the section Enzymes for fabric conditioning and processing, and food processing in Chapter 17; CLICK HERE to view now),
- ‘cellulase’ by Trichoderma reesei, some mutants of which produce up to 40 g l-1 total ‘cellulase’ activity of which half is the cellobiohydrolase known as CBH-l (see the section Digestion of lignocellulosic residues in Chapter 17; CLICK HERE to view now),
- penicillin by Penicillium chrysogenum, strain development by mutagenesis and strain selection of which is shown in Table 9 in the section Antibiotics and other pharmaceuticals in Chapter 17; CLICK HERE to view now).
- Natural genetic recombination; meaning classical ‘applied genetics’ involving cross-breeding to generate segregation and recombination of ‘desirable’ genes using the sexual cycle (although only a few of the fungi used in commercial industries reproduce sexually); the parasexual cycle; heterokaryosis or protoplast fusion; combined with artificial selection of the required combination of useful traits. This approach has been used successfully to improve productivity of glucoamylase by Aspergillus niger and exoglucanase by Trichoderma reesei. Generally speaking, mutagenesis and recombination strategies increase productivity by less than two-fold in a single step.
- Genetic manipulation; meaning the use of recombinant DNA technology to create a potentially unnatural fungal genotype that has commercially desirable characteristics.
We are concentrating on 21st century mycology in this book but before we go much further we must emphasise that recombinant DNA technology was developed in several model filamentous fungi more than a generation ago. A few noteworthy examples are:
- 1973: The first DNA-mediated transformation of a fungal species using genomic DNA without the use of vectors was carried out by Mishra & Tatum (1973), who transformed an inositol-requiring mutant strain of Neurospora crassa strain to inositol independence using DNA extracted from an inositol-independent strain.
- 1979: Case et al. (1979) developed an efficient transformation system for Neurospora crassa that used sphaeroplasts and a recombinant Escherichia coli plasmid carrying the N. crassa qa-2+ gene (which encodes the enzyme dehydroquinase).
- 1983: Ballance, Buxton & Turner (1983) performed the first auxotrophic marker transformation in Aspergillus nidulans when they relieved an auxotrophic requirement for uridine in a mutant strain of A. nidulans by transformation with a cloned segment of Neurospora crassa DNA containing the corresponding (= homologous) gene coding for orotidine-5′-phosphate decarboxylase.
- 1985 saw the successful transformation of a filamentous industrial fungus when Buxton, Gwynne & Davies (1985) transformed sphaeroplasts of a mutant of Aspergillus niger defective in ornithine transcarbamylase function with plasmids carrying a functional copy of the argB gene of A. nidulans, and Kelly & Hynes (1985) transformed A. niger, which cannot use acetamide as a nitrogen or carbon source, with the amdS (acetamidase) gene of A nidulans.
Recombinant proteins result from expression of genes which are introduced by recombinant DNA techniques. The product is described as being homologous protein where it results from expression of additional copies of a gene native to the species (for example, the transformation of the inositol-requiring mutant strain of Neurospora crassa mentioned immediately above). In contrast, the product is a heterologous protein when it results from expression of a non-native gene (for example, the A. nidulans acetamidase expressed in transformed A. niger mentioned above).
The basic strategy for improving protein productivity by recombinant DNA technology is outlined in the flow chart in Fig. 22.
|Fig. 22. Flowchart outlining the basic strategy for improving protein productivity by recombinant DNA technology.|
The initial isolation of the gene of interest may be done by direct complementation, in which the emphasis is on identifying a sequence that complements (that is, compensates for) the known defective phenotype in a test mutant. For example, an invertase-deficient mutant of Saccharomyces cerevisiae has been used to identify by complementation the corresponding invertase genes cloned from Neurospora crassa and Aspergillus niger. The most commonly employed method now, though, is by reverse genetics (Fig. 23) in which sequence information about the target protein and/or gene is used to design synthetic oligonucleotide probes that are then used to isolate the gene sequence from cDNA (will be free of signalling and intron sequences) or genomic DNA (will contain signalling and intron sequences) libraries.
|Fig. 23. Flowchart outlining the basic strategy for isolation of the gene of interest using recombinant DNA technology.|
Updated December 17, 2016