The first saprophytic cultivation of Claviceps on artificial nutritional media dates back to 1922 (Bonns 1922). The first attempt for the industrial production of ergot alkaloids was isolation of clavine alkaloids from submerged cultures of different Claviceps spp. (Abe and Yamatodani 1954; 1955; Abe et al. 1952; 1956). Later, conditions for saprophytic production of simple derivatives of lysergic acid were developed using different strains of C. paspali (Arcamone et al. 1960; 1961). It took only 5 years more when new isolate of C. purpurea was found to produce ergotamine under submerged conditions (Amici et al. 1966). Since that time all types of ergot alkaloids for direct use as therapeutic agents or precursors for the preparation of semisynthetic drugs can be obtained by fermentation.
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As with other fermentation processes the key to successful production of ergot alkaloids is in obtaining the proper strain of the fungus. There are three main processes used for the preparation of Claviceps strains for saprophytic culture. (a) Plating of plectenchymatic tissue from the surface sterilized sclerotia on an agar growth medium (Mantle 1969), (b) Plating of honeydew drops containing conidia formed at early stage of Claviceps infection (Janardhanan and Husain 1984; Pazoutova et al. 2002), and (c) Trapping of sexual ascospores ejected from fruiting bodies on germinated sclerotia. By this method monosporic culture can be obtained (Vasarhelyi et al. 1980).
The classical methods (selection pressure, mutagenesis, and recombination) used for the strain improvement are, to some extent, more complicated with Claviceps due to incomplete information on cell nucleus. Strains used for saprophytic cultivation might be heterokaryotic and homokaryotic (Didek-Brumec et al. 1991; Mantle and Nisbet 1976). Recently it was found that the number of chromosomes in C. purpurea is variable so that haploid as well as aneuploid nuclei may be encountered (Hiisgen et al. 1999). Mutagenesis followed by subsequent selection of strain is an important technique in increasing the yield of alkaloids (Didek-Brumec et al. 1987). An ergocristine producing C. purpurea strain showed 180-fold increase in alkaloid production after eight-step mutation-selection with different mutagens (Kobel and Sanglier 1978). Mutagenesis of sporulating strain results in monosporic isolates. To increase the mutation frequency the protoplasts prepared from spores of selected strains were used (Olasz et al. 1982; Zalai et al. 1990). More complicated situation is with mutagenesis of asporogenic strains. Hyphal fragments are rather unsuitable for mutagenesis due to the higher number of nuclei. Even protoplast formation from young mycelium and subsequent regeneration without any mutagenic treatment yielded strains with different properties (Schumann et al. 1987). Protoplast fusion technique is beginning to find useful applications either in producing improved mutant strains by intraspecific crosses or in formation of novel spectrum of products by interspecific hybrids (Socic and Gaberc-Porekar 1992). Relevant structural and regulatory genes of the alkaloid biosynthesis in C. purpurea form a cluster of about 50kbp in length (Tudzynski et al. 2001), therefore, isolation and cloning of the entire pathway to more rapidly growing fungus would be difficult.
5.2.4 Maintenance Improvement and Long-Term Preservation
Degeneration, loss of production capabilities, is a general problem of high-yielding strains of Claviceps (Kobel 1969). Conservation and systematically performed selection of the isolates is the only way to eliminate the biological effects given by the transfer of cultures, ageing, and other influences. Methods applied for long-term preservation were recently reviewed (Hunter-Cervera and Belt 1996). For preservation of sporulating cultures two main methods are applied: deepfreezing and maintaining of cultures on rye grains or agar slants placed in refrigerator. Nonsporulating strains due to higher sensitivity to conservation procedure are frequently preserved as cultures on the agar plates. The universal technique applied for preservation is keeping of lyophilized cultures and cultures frozen, in liquid nitrogen (Baumert et al. 1979).
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