that they were capable of secreting a range of enzymes and, thus, the strains were presumed to be modified in their secretion mechanisms. It is interesting to note that an improved secreter of a heterologous protein has been isolated using a traditional mutation and selection approach which has been miniaturized and automated.
In several cases the examination of randomly selected high-yielding mutants has indicated that their superiority may have been due to modifications of their control systems. Goulden and Chataway (1969) demonstrated that a mutant of P. chrysogenum, producing high levels of penicillin, was less sensitive to the control of acetohydroxyacid synthase by valine. Valine is one of the precursors of penicillin and its synthesis is controlled by its feedback inhibition of acetohydroxyacid synthase. Thus, removal of the control of valine synthesis may result in the production of higher valine levels and, hence, greater production of penicillin. Pruess and lohnson (1967) demonstrated that higher-yielding strains of P. chrysogenum also contained higher levels of acyltransferase, the enzyme which catalyses the addition of phenylacetic acid to 6-aminopenicillanic acid. Dulaney (1954) reported that the best producer of streptomycin amongst Streptomyces griseus mutants was auxotrophic for vitamin B12 and Demain (1973) claimed that this auxotrophic mutation was still a characteristic of production strains being used in 1969. Thus, it appears that some strains isolated by random selection, and overproducing secondary metabolites, are altered in ways similar to those strains isolated by directed selection techniques and overproducing primary metabolites.
There are many examples in the literature where a more directed selection approach has been adopted for the improvement of secondary metabolite producers.
phe techniques used include the isolation of auxotrophs, revertants and analogue-resistant mutants.
THt; ISOLATION OF AUXOTROPHIC MUTANTS
Although mutation of secondary metabolite producers to auxotrophy has resulted frequently in their producing lower yields, cases of improved productivity have also been demonstrated. For example, Alikhanian et al. (1959) investigated the tetracycline producing abilities of fifty-three auxotrophic mutants, all of which produced significantly less tetracycline than the parent strain in normal production medium. Supplementation of the medium with the growth requirement resulted in one mutant expressing productivity superior to that of the parent.
It is sometimes difficult to explain the precise reason for the effect of mutation to auxotrophy on the production of secondary metabolites, but in the majority of cases it has been demonstrated to be an effect on the secondary metabolic system rather than, simply, an effect on the growth of the organism. The simplest explanation for the deleterious effect on secondary metabolite yield is that the auxotroph is blocked in the biosynthesis of a precursor of the end product, for example, Polsinelli et al. (1965) demonstrated that aux-otrophs of Streptomyces antibioticus which required any of the precursors of actinomycin (isoleucine, valine or threonine) were poor producers of the antibiotic.
Many secondary metabolites may be considered as end products of branched pathways which also give rise to primary metabolites. Thus, a mutation to auxotrophy for the primary end product may also influence the production of the secondary product. In P. chryso-genum, lysine and penicillin share the same common biosynthetic route to a-aminoadipic acid, as shown in Fig. 3.25. This biosynthetic route may explain Bonner's (1947) observation that 25% of the lysine auxotrophs of P. chrysogenum he isolated could not produce penicillin. The role of lysine in the penicillin fermentation has also led to the investigation of lysine auxotrophs as potential superior penicillin producers. Demain (1957) demonstrated that lysine was inhibitory to the biosynthesis of pencillin. The explanation of this phenomenon is considered to be the inhibition of homoci-trate synthase by lysine resulting in the depletion of a-aminoadipic acid required for penicillin synthesis (Demain and Masurekar, 1974). It may be postulated that lysine auxotrophs blocked immediately after a-aminoadipic acid would produce higher levels of penicillin due to the diversion of the intermediate towards
a-Ketoglutarate + Acetyl-CoA
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