7-1.3.3 Rational Selection

It involves selecting an improved producer out of a very large population of progeny. For example, some antibiotics, notably penicillin and tetracyclines, are chelators of heavy metal ions. The more of these antibiotics an organism produces, the more resistant it will be to heavy metals in the medium. Thus, selection for mutants resistant to heavy metals was used in the improvement of the penicillin producers.

7-1.3.4 Cloning of the candidate genes  

Recombinant DNA technology can be used to introduce genes coding for antibiotic synthetases into producers of other antibiotics or into non-producing strains to obtain modified or hybrid antibiotics. The use of recombinant DNA technology in antibiotic improvement and discovery has been enhanced by the finding that some streptomycetes antibiotic biosynthetic pathways are coded by plasmid genes, eg. methylenomycin A. Even when the antibiotic biosynthetic pathway genes of streptomycetes are chromosomal, they appear to be clustered into operons which facilitate transfer of an entire pathway in a single manipulation.

The genes encoding individual enzymes of antibiotic biosynthesis which have already been cloned include those of the cephalosporin, clavulanic acid, prodigiosin, undecylprodigiosin, actinomycin, and candicidin pathways. The isopenicillin N synthetase ("CyClaSe") gene of Cephalosporium acremonium has been cloned in Escherichia coli and expressed at a level of 20% of total cell protein. Cyclase gene of Penicillium chrysogenum and Streptomyces clavuligerus has also been cloned in E.coli system. The expandase/ hydroxylase gene of C. acremonium has been cloned in E. coli. The protein accumulated as inclusion bodies in E. coli near to 15% of total cell protein