Fungal Genetics
Fungi possess strikingly different morphologies. They include large, fleshy, and often colorful mushrooms or toadstools, filamentous organisms only just visible to the naked eye, and single celled organisms such as yeast's. Moulds are important agents of decay. They also produce a large number of industrially important compounds like antibiotics (penicillin, griseofulvin, etc), organic acids (citric acid, gluconic acid, etc.), enzymes (alpha-amylases, lipase, etc.), traditional foods (softening and flavoring of cheese, shoyu soy sauce, etc.) and a number of other miscellaneous products (gibberellins, ergot alkaloids, steroid bioconversions). As late as 1974, the only widely applicable techniques for strain improvement were mutation, screening, and selection. This has proved dramatically successful in improving penicillin production. However, this deflected attempts to employ a more sophisticated approach to genetic manipulation. The study of fungal genetics has recently changed beyond all recognition.
The natural genetic variation present in fungal species has been characterized using molecular methods such as electrophoretic karyotyping, restriction fragment length polymorphism, DNA finger printing, and DNA sequence comparisons. The causes for the variation include chromosomal polymorphism, changes in repetitive DNA, transposons, virus like elements, and mitochondrial plasmids.
Genetic recombination occurs naturally in many fungi. Many industrially important fungi such as Aspergilli and Penicillia lack sexuality, so in these species parasexual systems (cycles) provide the basis for genetic study and breeding programmes. The parasexual cycle is a series of events that can be induced when two genetically different strains are grown together in the laboratory. A heterokaryon, which is mycelium with two different nuclei derived from two different haploid strains, is produced by the fusion of hyphae. Increased penicillin titer in the haploid progeny of parasexual crosses has been achieved in Penicillium chrysogenum. A more direct approach has been developed using protoplasts. These are isolated from vegetative cells of fungi or yeasts by removing the cell wall by digestion using a cell wall degrading enzyme. Protoplasts from the two strains can be fused by treatment with polyethylene glycol. Protoplast fusion in fungi initiates the parasexual cycle, resulting in the formation of diploidy and mitotic recombination and segregation. A selection procedure to screen such fusants is done using genetic markers. A good example of applying this technique is the fusion of a fast growing but poor glucoamylase producer with a slow growing but excellent producer of glucoamylase. The desired result will be a strain, which is both fast growing and an excellent producer of enzyme.
The realization that transformation of genetic material into fungi can occur came with the discovery that yeasts like Saccharomyces cerevisiae and filamentous fungi like Podospora anserine contain plasmids. Currently transformation technology is largely based on the use of Neurospora crassa and Aspergillus nidulans, although methods for use in filamentous organisms are being evolved. The protocols used in transformation of filamentous fungi involve cloning the desired gene into the plasmid from E.coli or a plasmid constructed from genetic material from E.coli and Saccharomyces cerevisiae. Protoplasts from the recipient strains are then formed and mixed with the plasmid. After incubating for a short time to allow for the uptake of the plasmid DNA, the protoplasts are allowed to regenerate and the cells are screened for the presence of the specific marker.
The application of recombinant DNA to yeast's and filamentous fungi has opened up new possibilities in relation to the construction of highly productive strains. The filamentous fungi are now established as potent host organisms for the production of heterologous proteins. This is particularly useful as expression of specific proteins can reach relatively high levels. Using Aspergillus as a host for reproduction as led to the production of many recombinant products like human therapeutic proteins, including growth factors, cytokines and hormones. While expression can be good in E.coli, lack of posttranslational modifications has limited their usage. The use of Saccharomyces species has not been that successful for the production of extracellular proteins. Most of the initial advances for the production of heterologous proteins has been with filamentous fungi have been with Aspergillus nidulans. Although this organism is not of industrial importance it is nevertheless genetically well characterized. Besides this organism has secretion signals which results in recombinant proteins being identical to mammalian cells. This allows the product from such systems to be used safely in human therapy. Other systems that have been used include Pichia and Trichoderma have been for the production of a large number of useful products. Now that the complete genome of S. cerevisiae has been deciphered, and more fungi genomes in the pipe line, an even better understanding of fungal genetics is certain.
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