Quv

Mutation treatment

Survivors of the mutation treatment growing on agar medium

200 colonies removed and each suspended in sterile water

Survivors of the mutation treatment growing on agar medium

200 colonies removed and each suspended in sterile water

3 stock slopes of each isolate

3 stock slopes of each isolate

Spore suspension of each isolate used to inoculate an inoculum development flask

Each inoculum flask used to inoculate a flask of production medium

The best 50 producers re-tested in quadruplicate

Spore suspension of each isolate used to inoculate an inoculum development flask

Each inoculum flask used to inoculate a flask of production medium v Best 4 conserved and re-mutated Fig. 3.24. A strain-improvement programme for a secondary metabolite producing culture (Davies, 1964).

cal variant may be a superior producer, it might require considerable fermentation development to materialize the increased production. Also, Alikhanian (1962) claimed that the vast majority of morphological mutants of the antibiotic producing actinomycetes tested were inferior producers. The most common type of shake flask programme quoted is similar to that of Davies where the choice of colonies is random. However, there are now many reports of screens which miniaturize the procedure for the improvement of secondary metabolite producing strains. Such miniaturized systems are designed to enable the productivity of all (or a significant proportion) of the survivors to be assessed which should eliminate (or reduce) the problem of choice of colonies for assessment and increase the throughput of cultures.

The basis of the miniaturized techniques is to grow the survivors of the mutation treatment either in a very low volume of liquid medium or on solidified (agar) medium. If the product is an antibiotic, the agar-grown colonies may be overlayed with an indicator organism sensitive to the antibiotic produced, allowing assay to be done in situ. The level of antibiotic is assessed by the degree of inhibition of the overlayed indicator. The system is simple to apply to strains producing low levels of antibiotic but must be modified to allow the screening of high producers where very large zones of inhibition would be obtained. Also a system should be used to free the superior producers from contaminating indicator organisms. Dulaney and Dulaney (1967) used overlay techniques in the isolation of mutants producing chlortetracycline. Mutated spores were cultured on an agar medium in petri dishes for 6 days and then covered with pieces of sterile cellophane. An overlay of agar containing the indicator organism was then added and the plates incubated overnight. The mutant colonies were kept free from contamination by the cellophane and the size of the inhibition zone could be controlled by the depth of the base layer, the age of the colonies when over-layed, the depth of the overlay and the temperature of incubation. The system was calibrated initially such that a single colony of a non-mutated strain would not produce an inhibition zone but that two such adjacent colonies would. In practice, the size of the inhibition zone was controlled by the depth of the overlay. Dulaney and Dulaney obtained a far greater enrichment in the number of desired phenotypes by the overlay technique than by random selection and testing in liquid medium.

Ichikawa et al. (1971) screened for the increased production of the antibiotic kasugamycin by a Strep-tomyces sp., using a miniaturized technique termed the agar piece method. In order to prevent interference between colonies, mutated spores were grown on plugs of agar which were then placed on assay plates containing agar seeded with the indicator organism, levels of the antibiotic being determined by the size of inhibition zones. By combining this technique with a medium improvement programme the authors obtained a tenfold increase in productivity. Ichikawa's method was modified by Ditchburn et al. (1974) for the isolation of Aspergillus nidulans mutants synthesizing improved levels of penicillin. These workers obtained promising results using the technique and claimed that its potential for the recovery of higher-yielding mutants, for a given expenditure of labour time, was greater than the shake-flask method. The level of production of penicillin by A. nidulans is very small compared with P. chrysogenum and the technique would have to be modified considerably for commercial use.

Ball and McConagle (1978) developed a miniaturized technique suitable for the assay of penicillin production by 'high-yielding' strains of P. chrysogenum (producing up to 6000 units cm"3). These workers highlighted the design of the solidified medium as a critical factor in the optimization of the method. They claimed that the growth of a colony on agar-solidified medium would be unlikely to modify the medium to the same extent as the growth of the organism on the same medium in submerged liquid culture. Thus, the nutrient-limiting conditions that favour the onset of antibiotic production might not be achieved by the growth of the culture on solid medium which would not allow the full production potential of the culture to be detected. However, nutrient-limiting conditions were achieved in the solidified medium by omitting the main carbon source and reducing the corn steep-liquor content. Mutated spores were plated over the surface of petri dishes of the nutrient limited medium such that ten to twenty colonies developed per plate. The time of incubation of the plates was not quoted but "when the colonies were of a size suitable for accurate measurement" a pasteurized spore suspension of B. subtilis containing 0.16 units cm~3 of penicillinase (to limit the size of the inhibition zones) was dispersed over the surface of the dish. The plates were then incubated for 18 to 24 hours and the inhibition zones examined. Suitable colonies were freed of contaminating B. subtilis by culturing on nutrient agar containing sodium penicillin and streptomycin sulphate. The use of this technique enabled three operators to scan 15,000 survivors from ultraviolet irradiated spore populations in 3 months.

The major disadvantages of the miniaturized solidi-

fled medium technique approach is that productivity expressed on the solid medium may not be expressed in subsequent liquid culture and conversely, colonies not showing activity on solid media may be highly productive in liquid medium. Despite these limitations the above workers have demonstrated that the approach has considerable merit and Ball (1978) claimed that the increase in throughput may be as much as 20 times that of a conventional shake-flask programme. Some recent work by Bushell's group (Pickup et al., 1993) provides an interesting insight into the problem of certain strep-tomycete isolates producing a secondary metabolite on agar but not in liquid culture. It is important to appreciate that the strains were natural isolates and not the survivors of mutation treatments but the conclusions do have relevance to mutant development. Working on the premise that non-production in liquid medium may be due to a more fragmented morphology these strains were subjected to a filtration enrichment procedure. Liquid cultures were filtered through a linen filter and the filtrate and retentate mycelium re-cultured in fresh medium. The procedure was then repeated sequentially. The enriched retentate mycelium of several isolates which were previously unable to produce antibiotic in liquid culture gave rise to stable filamentous types synthesizing in liquid medium. Although this approach could not be used routinely in a high throughput mutant screen it may be applied to a few high-producing strains which have not fulfilled their promise (detected in an agar screen) in liquid media.

An excellent example of a miniaturized screening programme is given by the work of Dunn-Coleman et al. (1991) of Genencor International. These workers developed a low-volume liquid medium system to isolate mutants of Aspergillus niger var. awamori capable of improved secretion of bovine chymosin. The Aspergillus strain had been constructed using recombinant DNA technology and this construction will be considered in a later section of this Chapter. Mutated spore suspensions were diluted and inoculated into 96-well microtitre plates using robots. The dilutions were such that each well should have contained one viable spore. The plates were incubated in a static incubator and then harvested using a robotic pipetting station and assayed for product. Typically, 50-60,000 mutated viable spores were assessed in each screen. The most promising 10-50 strains from the miniaturized screen were then tested in shake flask culture. The best producer from the shake-flask screen was then used as the starter for a further round of mutagenesis. The results of seven rounds of mutation and selection are shown in Table 3.6. Investigation of the improved strains showed

Table 3.6. Chymosin production by NTG mutated strains of As. pergillus niger var awamori (Dunn-Coleman etal., 1991)

Chymosin concentration (/xg cm-3)

Rounds of NTG

mutagenesis and Microtitre Shake flask recurrent selection plate mutagenesis and Microtitre Shake flask recurrent selection plate

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