The Isolation Of Mutants Which Do Not Produce Feedback Inhibitors Or Repressors

Mutants which do not produce certain feedback inhibitors or repressors may be useful for the production of intermediates of unbranched pathways and intermediates and end products of branched pathways. Demain (1972) presented several 'blue-prints' of hypothetical mutants producing intermediates and end products of biosynthetic pathways and these are illustrated in Fig. 3.12. The mutants illustrated in Fig. 3.12

do not produce some of the inhibitors or repressors of the pathways considered and, thus, the control of the pathway is lifted, but, because the control factors are also essential for growth, they must be incorporated into the medium at concentrations which will allow growth to proceed but will not evoke the normal control reactions.

In the case of Fig. 3.12(1) the unbranched pathway is normally controlled by feedback inhibition or repression of the first enzyme of the pathway by the end product, E. However, the organism represented in Fig. 3.12(1) is auxotrophic for E due to the inability to convert C to D so that control of the pathway is lifted and C will be accumulated provided that E is included in the medium at a level sufficient to maintain growth but insufficient to cause inhibition or repression.

Figure 3.12(2) is a branched pathway controlled by the concerted inhibition of the first enzyme in the pathway by the combined effects of E and G. The mutant illustrated is auxotrophic for E due to an inability to convert C to D, resulting in the removal of the concerted control of the first enzyme. Provided that E is included in the medium at a level sufficient to allow growth but insufficient to cause inhibition then C will be accumulated due to the control of the end product G on the conversion of C to F. The example shown in Fig. 3.12(3) is similar to that in Fig. 3.12(2) except that it is a double auxotroph and requires the feeding of both E and G. Figure 3.12(4) is, again, the same pathway and illustrates another double mutant with the deletion for the production of G occurring between F and G, resulting in the accumulation of F.

Figure 3.12(5) illustrates the accumulation of an end product of a branched pathway which is normally controlled by the feedback inhibition of the first enzyme in the pathway by the concerted effects of E and I. The mutant illustrated is auxotrophic for I and G due to an inability to convert C to F and, thus, provided G and I are supplied in quantities which will satisfy growth requirements without causing inhibition, the end product, E, will be accumulated.

All the hypothetical examples discussed above are auxotrophic mutants and, under certain circumstances, may accumulate relatively high concentrations of intermediates or end products. Therefore, the isolation of auxotrophic mutants may result in the isolation of high-producing strains, provided that the mutation for auxotrophy occurs at the correct site, e.g. between C and D in Figs 3.12(1) and (2). The recovery of aux-otrophs is a simpler process than is the recovery of high producers, as such, so that the best approach is to

Feed limiting concentration of E

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