Feed limiting concentration of E and G

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Site of auxotrophic mutation Feedback regulation Overproduced product

Fro. 3.12. Overproduction of primarymetabolites by decreasing the concentration of a repressing or inhibiting end product, auxotrophicmutations;----.feedback regulation; -ยป overproduced product (Demain, 1972).

Site of design a procedure to select relevant auxotrophs from the survivors of a mutation and subsequently screen the selected auxotrophs for productivity. Productive strains, amongst the auxotrophs may be detected by over-layering colonies of the mutants with agar suspensions of bacteria auxotrophic for the required product. The high-producing mutants may be identified by the growth of the overlay around the producer. The most commonly used methods for the recovery of auxotrophic mutants are the use of some form of enrichment culture or the use of a technique to visually identify the mutants.

The enrichment processes employed are based on the provision of conditions which adversely affect the prototrophic cells but do not damage the auxotrophs. Such conditions may be achieved by exposing the population, in minimal medium, to an antimicrobial agent which only affects dividing cells which should result in the death of the growing prototrophs but the survival of the non-growing auxotrophs. Several techniques have been developed using different antimicrobials suitable for use with a range of micro-organisms.

Davis (1949) developed an enrichment technique utilizing penicillin as the inhibitory agent. The survivors of a mutation treatment were first cultured in complete medium, harvested by centrifugation, washed and re-suspended in minimal medium plus penicillin. Only the growing prototrophic cells were susceptible to the penicillin and the non-growing auxotrophs survived. The cells were harvested by centrifugation, washed (to remove the penicillin and products released from lysed cells) and resuspended in complete medium to allow the growth of the auxotrophs, which could then be purified on solid medium. The nature of the auxotrophs isolated may be determined by the design of the so-called complete medium; if only one addition is made to the minimal medium then mutants auxotrophic for the additive should be isolated.

Abe (1972) described the use of Davis' technique to isolate auxotrophic mutants of the glutamic acid producing organism C. glutamicum. The procedure is outlined in Fig. 3.13.

Advantage has also been taken of the fact that the ungerminated spores of some organisms are more resistant to certain compounds than are the germinated spores. Thus, by culturing mutated spores in minimal

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