» Biosynthetic route ---- Feedback inhibition
Fig. 3.25. Biosynthesis of benzyl penicillin and lysine in Pénicillium chrysogenum.
penicillin synthesis and the removal of any control of homocitrate synthase by endogenous lysine. O'Sullivan and Pirt (1973) investigated the production of penicillin by lysine auxotrophs of P. chrysogenum in continuous culture but were unable to demonstrate improved productivity in a range of lysine-supplemented media. Luengo et al. (1979) examined the lysine regulation of penicillin biosynthesis in low-producing and industrial strains of P. chrysogenum. The industrial strain was capable of producing up to 12,000 units cm*3 penicillin. These workers demonstrated that although the onset of penicillin synthesis in the industrial strain was less sensitive to lysine than in the low-producing strain, the empirical industrial selection procedures had not completely removed the mechanisms of lysine regulation of penicillin biosynthesis. Luengo et al. expressed the possibility that overproducers of penicillin may be obtained by the selection of mutants to lysine regulation. However, it should be noted that the industrial strain employed was a representative of only one series of penicillin producers and that other series may have already been modified with respect to the regulatory effects of lysine. Nevertheless, this study does indicate the possibility that selection of strains resistant to lysine control may be overproducers of penicillin.
It is far more difficult to explain the effect of aux-otrophy for factors not associated with the biosynthesis of the secondary metabolite. Polsinelli et al. (1965) demonstrated that seven out of twenty-seven aux-otrophs of S. griseus produced more actinomycin than did the prototrophic parent. Dulaney and Dulaney (1967) demonstrated increased chlortetracycline yields in eight out of eleven auxotrophs of S. viridifaciens when grown in supplemented media. In neither of these cases were the auxotrophic requirements directly involved in the biosynthesis of the secondary metabolites. Demain (1973) put forward two possible explanations to attempt to account for the behaviour of such auxotrophs. The first explanation is that the auxotrophic factors were involved in 'cross-pathway' regulation with the secondary metabolite or its precursors. Demain quoted several examples of cross-pathway regulation in primary metabolism where the activity of one pathway is affected by the product of an apparently unrelated sequence. The alternative explanation is that the effect on secondary metabolism is not due to the auxotrophy but to a second mutation accompanying the auxotrophy, i.e. a double mutation. Demain cited two attempts to determine whether the effects of auxotrophy on secondary metabolism were due to double mutations or to the auxotrophy. MacDonald et al. (1963) reverted a low-producing thiosulphate-requiring mutant of P. chrysogenum to thiosulphate independence and examined penicillin productivity by the revertants. Approximately half of the revertants re-acquired their 'grandparents' production level, whereas the other half retained their poor productivity. Polsinelli et al. (1965) reverted five isoleucine-valine auxotrophs of S. antibi-oticus (which also produced low levels of actinomycin compared with the parent strain) to prototrophy and discovered that some were returned to normal production and others to higher production levels than the 'grandparent'. Thus, in the case of Polsinelli et al.'s mutants it is unlikely that the effect on secondary metabolism was due to a double mutation, but it is possible that this was the case for some of MacDonald et al.'s strains. However, it should be remembered that both these groups of auxotrophs were poor secondary metabolite producers blocked in routes directly involved with the secondary biosynthetic pathway. It may be more relevant to examine the nature of auxotrophic strains blocked in apparently unrelated pathways and produce improved levels of the secondary metabolite. It is not possible to say whether any of the auxotrophic mutants previously discussed produced superior levels of the secondary metabolite as a result of double mutations, but it may be possible to exploit this possibility in the future.
The treatment of bacterial cells with nitrosoguani-dine (NTG) has been demonstrated to result in clusters of mutations around the replicating fork of the chromosome (Guerola et al., 1971). Thus, if one of the mutations were selectable (for example, auxotrophy) it may be possible to isolate a strain containing the selectable mutation along with other non-selectable mutations which map close by. The efficient application of this technique would depend on the accurate mapping of the gene involved in producing the secondary metabolite so that neighbouring mutations may be selected. This technique may be valuable for the selection of mutants of the bacilli and streptomycetes producing high levels of antibiotics where mutations affecting synthesis may be mapped for each biosynthetic step. Co-mutation by NTG may then be followed by selection for changes to genes adjacent to those loci known to be involved in production of the particular secondary metabolite.
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