7-2.1 Introduction

Genetic manipulation of biosynthetic pathways is a useful method for producing analogs of complex bioactive metabolites. Reconstruction of biosynthetic gene clusters in E. coli could be done for rapid heterologous production of natural products and genetic manipulation of their biosynthetic pathways. But the real challenge lies in the suitable expression of proteins in recombinant microbe system. To obtain enzymes, antibiotics in its native, functional folded structure is the ultimate bottleneck of recombinant technology.

7-2.2 Production of Antibiotics

Antibiotics are small molecular weight compounds that inhibit or kill microorganisms at low concentrations. Antibiotics are produced by various bacteria, actinomycetes and fungi such as Bacillus, Streptomyces, and Penicillium. The significance of antibiotic production in micro organisms is still unclear which may be for ecological adaptation for the organism in nature.

7-2.3 Engineering Escherichia Coli to Produce Non- Ribosomal Peptide Antibiotics

• A monocistronic reconstituted form of the ecm gene (extra cellular matrix gene) cluster from Streptomyces lasaliensis was cloned and expressed in E. coli that directs the biosynthesis of the anti- tumor non- ribosomal peptide echinomycin. Biosynthetic gene function was examined by constructing a set of expression plasmids containing only 15 of the 16 genes required for echinomycin production. The ecm18 gene cassette encoding a methyltransferase was omitted from one of the plasmids. Expression of the reconstructed cluster in E. coli results in production of echinomycin (red), whereas expression of the reconstructed cluster minus the ecm 18 gene results in production of triostin A (blue). It was confirmed that Ecm18 functions as a S-adenosylmethionine dependent methyltransferases (SAM-MTs) that catalyses the conversion of triostin A to echinomycin in vitro using the purified recombinant enzyme.