A REGULATORY GENE (CCAR) REQUIRED FOR CEPHAMYCIN AND CLAVULANIC ACID PRODUCTION IN STREPTOMYCES-CLAVULIGERUS - AMPLIFICATION RESULTS IN OVERPRODUCTION OF BOTH BETA-LACTAM COMPOUNDS

A regulatory gene (ccaR), located within the cephamycin gene cluster o f Streptomyces clavuligerus, is linked to a gene (blp) encoding a prot ein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. cla vuligerus. The ccaR-encoded protein resembles the ActII-ORB4, RedD, Af sR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs, Disruption of ccaR by targeted double rec ombination resulted in the loss of the ability to synthesize cephamyci n and clavulanic acid. Complementation of the disrupted mutant with cc aR restored production of both secondary metabolites. ccaR was express ed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cul tures (preceding the phase of antibiotic accumulation), but no transcr ipt hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resu lted in a two- to threefold increase in the production of cephamycin a nd clavulanic acid.