CLONING, SEQUENCING AND DISRUPTION OF A GENE FROM STREPTOMYCES-CLAVULIGERUS INVOLVED IN CLAVULANIC ACID BIOSYNTHESIS

During the purification of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-v aline synthetase (ACVS) from Streptomyces clavuligerus us, a small pro tein (CLA) involved in clavulanic acid production co-purified with ACV S. A 24-mer mixed-DNA probe based on the N-terminal amino-acid sequenc e of CLA was used to isolate the corresponding gene (cia), located nea r one end of the known cluster of penicillin and cephamycin biosynthet ic genes, 5.7 kb downstream from the pcbC gene which encodes isopenici llin-N synthase. The sequence of cia would encode a protein of 313 aa with a high degree of similarity to amidinohydrolase enzymes. The cla gene is located immediately upstream from the previously described cla vaminate synthase 2-encoding gene (cs2), and cia homologs were only pr esent in streptomycetes which produced clavam compounds. Replacement o f cla with a disrupted copy of the gene blocked the production of clav ulanic acid in starch asparagine medium (SA).