The clavulanic acid biosynthetic cluster of Streptomyces clavuligerus: genetic organization of the region upstream of the car gene

Área de Biotecnología, Antibióticos SA, Avenida de Antibióticos 59-61, 24009 León, Spain 1 Área de Microbiología, Facultad de Biología, Universidad de León, 24071 León, Spain 2 Author for correspondence: José L. Barredo. Tel: +34 987 895826. Fax: +34 987 895986. e-mail: jbarredo{at}antibioticos.it The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 12·1 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed: ORF10–11, ORF12–13 and ORF15–16–17–18. Potential transcriptional terminators were identified downstream of ORF11 ( fd ) and ORF15. Targeted disruption of ORF10 ( cyp ) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10–14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid. Keywords: antibiotic biosynthesis, gene cluster, cytochrome P450, ferredoxin, PBP Abbreviations: CA, clavulanic acid; PBP, penicillin-binding protein The GenBank accession number for the 12162 bp sequence reported in this paper is AY034175 .