Protoplasts are cells devoid of their cell walls and may be prepared by subjecting cells to the action of wall degrading enzymes in isotonic solutions. Protoplasts may regenerate their cell walls and are then capable of growth as normal cells. Cell fusion, followed by nuclear fusion, may occur between protoplasts of strains which would otherwise not fuse and the resulting fused protoplast may regenerate a cell wall and grow as a normal cell. Thus, protoplasts may be used to overcome some recombination barriers. Protoplast fusion has been demonstrated in a large number of industrially important organisms including Streptomyces spp. (Hopwood et al., 1977), Bacillus spp. (Fodor and Alfoldi, 1976), corynebacteria (Karasawa et al., 1986), filamentous fungi (Ferenczy et al., 1974) and yeasts (Sipiczki and Ferenczy, 1977).
Fusion of fungal protoplasts appears to be an excellent technique to obtain heterokaryons between strains where conventional techniques have failed, or, indeed, as the method of choice. Thus, this approach has allowed the use of the parasexual cycle for breeding purposes in situations where it had not been previously possible. This situation is illustrated by the work of Peberdy et al. (1977) who succeeded in obtaining heterokaryons between P. chrysogenum and P. cyaneoful-vum and demonstrated the formation of diploids which gave rise to recombinants after treatment with /j-fluo-rophenylalanine or benomyl. Although it has been claimed that P. chrysogenum and P. cyaneofulvum are not different species of Pénicillium (Samson et al., 1977), Peberdy et al. still demonstrated that protoplast fusion could be successful where conventional techniques had failed.
A demonstration of the use of protoplast fusion for an industrial fungus is provided by the work of Hamlyn and Ball (1979) on the cephalosporin producer, C. acremonium. These workers compared the effectiveness of conventional techniques of obtaining nuclear fusion between strains of C. acremonium with the protoplast fusion technique. The results from conventional techniques suggested that nuclear fusion was difficult to achieve. Electron microscopic examination of fused protoplasts indicated that up to 1% underwent immediate nuclear fusion. Recombinants were obtained in both sister and divergent crosses. A cross between an asporulating, slow-growing strain with a sporulating fast-growing strain which only produced one-third of the cephalosporin level of the first strain eventually resulted in the isolation of a recombinant which combined the desirable properties of both strains, i.e. a strain which demonstrated good sporulation, a high growth rate and produced 40% more antibiotic than the higher-yielding parent. Chang et al. (1982) utilized protoplast fusion to combine the desirable qualities of two strains of Pénicillium chrysogenum. Protoplasts from two strains, differing in colony morphology and the abilities to produce penicillin V (the desired product)
and OH-V penicillin (an undesirable product), were fused, followed by plating on a non-selective medium. Out of 100 stable colonies which were scored, two possessed the desirable morphology, high penicillin V and low OH-V penicillin productivities.
Lein (1986) reported the penicillin strain improvement programme adopted by Panlabs, Inc. This programme included random and directed selection as well as protoplast fusion. Table 3.7 illustrates the properties of the two strains used in a protoplast fusion and one of the recombinants selected. To avoid any adverse effects no selective genetic markers were used and the regenerated colonies were screened on the basis of colony morphology and spore colour. A total of 238 colonies judged to be recombinants were screened for penicillin V production and the culture with the best combination of properties is shown in the table. Thus, the desirable characteristics of each strain were combined in the recombinant.
Protoplasts are also useful in the filamentous fungi for manipulations other than cell fusion. Rowlands (1992) suggested that they may be used in mutagenesis of non-sporulating fungi. Spores are the cells of choice for the mutagenesis of filamentous fungi but this is obviously impossible for non-sporulating strains. Mycelial fragments may be used but these will be multinucleate and very high mutagen doses are required. Although some protoplasts will be non-nucleate or multi-nucleate at least some will be uninucleate which will express any modified genes after mutation. Also, protoplasts will take up DNA in in vitro genetic manipulation experiments and this aspect will be discussed in a later section of this chapter.
Recombination can take place between acti-nomycetes by conjugation (Hopwood, 1976) and phage transduction (Studdard, 1979). However, both these mechanisms involve the transfer of only small regions of the bacterial chromosome. Furthermore, the low frequencies at which recombination occurs necessitate the use of selectable genetic markers such as auxotro-
phy or antibiotic resistance. The introduction of such markers is time-consuming but also they can detrimentally affect the synthetic capacity of the organism. Protoplast fusion has particular advantages over conjugation in that the technique involves the participation of the entire genome in recombination. Also, Hopwood (1979) has developed techniques which have resulted in the recovery of a very high proportion of recombinants from the fusion products of Streptomyces coelicolor protoplasts. By subjecting protoplasts to an exposure of ultraviolet light, sufficient to kill 99% of them prior to fusion, Hopwood has claimed a tenfold increase in recombinant detection for strains normally giving a low yield of recombinants (1%) and a doubling of the recombination frequency for a cross normally yielding 20% recombinants. Such yields of recombinants means that they would be detectable by simply screening a random proportion of the progeny of a protoplast fusion and the use of selectable markers to 'force' out the recombinants would not be necessary. Examples of the application of the technique to actinomycete strain improvement include cephamycin C yield enhancement in Nocardia lactamdurans (Wesseling, 1982) and the improvement of lignin degradation in Streptomyces viri-dosporus (Petty and Crawford, 1984).
Protoplast fusion has also been applied to the improvement of amino acid producing strains. Karasawa et al. (1986) used the technique to improve the fermentation rates of lysine producers developed using repeated mutation and directed selection. Such strains were good lysine producers but showed low glucose consumption and growth rates, undesirable features which had been inadvertently introduced during the selection programme. A protoplast fusion was performed between the lysine producer and a fast growing strain and a fusant was isolated displaying the desirable characteristics of high lysine production and high glucose consumption rate resulting in a much faster fermentation. The same authors used protoplast fusion to produce a superior threonine producing Brevibacterium
Table 3.7. The me of protoplast fusion for the improvement of a pencillin V producer (Lein, 1986)
Characteristic Parent A Parent B Best recombinant
Germination frequency (%) 99 40 49
Colour of sporulating colonies Green Pale green Deep green
Seed growth Good Poor Good
Phenylacetic oxidation Yes No No lactofermentum strain. Lysine auxotrophy was introduced into a threonine and lysine overproducer by fusing it with a lysine auxotroph — the recombinant produced higher levels of threonine due to its lysine auxotrophy.
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