Mutasynthesis in Streptomyces for production of novel calcium-dependent antibiotics

The calcium-dependent antibiotic (CDA) is a lipopeptide synthesised non-ribosomally and produced by Streptomyces coelicolor A3(2). CDA contains several non-proteinogenic amino acid residues. Hydroxyphenylglycine (4-HPG) is one of the unusual amino acids in the structure of the CDA and vancomycin groups of antibiotics. For the members of the vancomycin group of antibiotics, the 4-HPG residue plays crucial roles in the structure and function of the final glycopeptide antibiotic.To reveal the putative biosynthetic pathway of this amino acid in CDA, a standard "double crossover replacement strategy" was used to delete 4-hydroxymandelic acid synthase (4-HMAS, encoded by hpd) from different strains of S. coelicolor, using the delivery plasmid pZMH3. An in frame-deletion removed the hpd gene from the 4-HPG operon and generated null mutations in S. coelicolor MT1110 and 2377 (S. coelicolor MT1110Δhpd and S. coelicolor 2377Δhpd). The deleted regions in both strains were confirmed initially using PCR and finally by Southern analysis. There was no CDA production in the disrupted strains. Plates containing a gradient of hydroxymandelic acid were used to restore CDA production in both S. coelicolor MT1110Δhpd. Exogenous supply of 4- hydroxyl phenylglyoxylate and 4-hydroxyphenylglycine re-established CDA production by the hpd mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). The high level of calcium dependent antibiotic activity was detected in these novel antibiotics.