The Isolation Of Revertant Mutants

As discussed in the section on primary metabolites, a mutant may revert to the phenotype of its 'parent', but the genotype of the revertant may not, necessarily, be the same as the original 'parent'. Some revertant aux-otrophs have been demonstrated to accumulate primary metabolites (Shiio and Sano, 1969) and attempts have been made to apply the technique to the isolation of mutants overproducing secondary metabolites. Two approaches have been used in the field of secondary metabolites with respect to the isolation of revertants:

(i) The isolation of revertants of mutants auxotrophic for primary metabolites which may influence the production of a secondary metabolite.

(ii) The reversion of mutants which have lost the ability to produce the secondary metabolite

(i) The isolation of revertants of mutants auxotrophic for primary metabolites which may influence the production of a secondary metabolite

As previously discussed, it is difficult to account for some of the effects of auxotrophy on secondary metabolism and it appears that some may be due to as yet unresolved cross-pathway phenomena and others due to the expression of other mutations associated with the auxotrophy. Similarly, revertant mutants may affect secondary metabolism in a number of ways — direct effects on the pathway, cross-pathway effects and effects due to mutations other than the detected reversion.

Dulaney and Dulaney (1967) investigated the tetracycline productivities of a population of prototroph revertants of S. viridifaciens derived from each of five auxotrophs. These workers predicted that some revertants may be productive due to direct influence of the mutations on tetracycline biosynthesis but that others may be so because they contained other lesions. Superior producers were obtained in all the prototroph-re-

vertant populations apart from those derived from a homocysteine auxotroph. However, the frequency of the occurrence of the superior producers was similar to that obtained by the random selection of the survivors of a mutation treatment in all but one of the populations. The exceptional population was the revertants of a methionine auxotroph, 98% of which produced between 1.2 and 3.2 times as much tetracycline as the original prototrophic culture. A possible explanation of the very favourable titres of the population may be the role of methionine as the methyl donor in tetracycline biosynthesis and that methionine availability limited the production of the secondary metabolite in the original prototroph. However, addition of exogenous methionine to the prototroph did not result in superior productivity.

Polsinelli et al. (1965) also demonstrated that reversion of five mutants of an actinomycin producing strain of S. antibioticus blocked in the isoleucine-valine pathway resulted in the isolation of some superior mutants. Godfrey (1973) reported that the reversion of a cysteine auxotrophic mutant of S. lipmanii resulted in improved production of cephamycin.

These studies provide promising evidence that the selection of revertants of auxotrophs of primary metabolites involved in secondary metabolism may yield a high number of productive mutants.

(ii) The isolation of revertants of mutants which have lost the ability to produce the secondary metabolite

The reversion of non-producing strains may result in the detection of a high-producing mutant as that mutant would have undergone at least two mutations associated with the production of the secondary metabolite. Dulaney and Dulaney (1967) plated the progeny of a mutation of a non-producing strain of S. viridifaciens onto solidified production medium and screened for superior tetracycline producers by an overlay technique. A mutant was isolated which produced nine times the tetracycline yield of the original 'parent'. The major difficulty inherent in this technique is that non-producing mutants of high-yielding strains may be incapable of being reverted due to extreme deficiencies in their metabolism. Indeed, Rowlands (1992) suggested that strains which are non (or low) producers and illustrate other effects such as poor growth and sporulation are best discarded, but that revertant mutants not showing pleiotropic effects are perhaps more likely to possess genuine increases in biosynthetic activity than those produced by any other technique, having been mutated twice in genes directly affecting product formation.

The use of recombination systems for the improvement of industrial micro-organisms

Hopwood (1979) defined recombination, in its broadest sense, as "any process which helps to generate new combinations of genes that were originally present in different individuals". The use of recombination mechanisms for the improvement of industrial strains has increased significantly due to the developments in recombinant DNA technology and the necessity to develop new methods of strain improvement as the returns generated from mutation and selection programmes decreased. However, it should be appreciated that mutation and selection techniques are frequently used in association with recombination systems in a strain improvement programme. The parasexual cycle in the filamentous fungi has been applied to strain development as have protoplast fusion techniques in a wide range of micro-organisms.

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