Cephalosporin formation by cell-free extracts from Streptomyces clavuligerus

Cell-free extracts of Streptomyces clavuligerus convert delta-L-(alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) into an antibiotic product which is 30 approximately 50% penicillinase-insensitive. Thin-layer chromatography resolves this antibiotic product into one major penicillinase-sensitive component and one major and one minor penicillinase-resistant component. The major and minor penicillinase-resistant antibiotics co-chromatograph with deacetoxycephalosporin C and deacetylcephalosporin C, respectively. Ring expansion of a penicillin intermediate, as evidenced by the production of penicillinase-resistant antibiotic, shows an absolute requirement for alpha-ketoglutarate, while ATP, K+ and Mg2+ have lesser effects. Ring expansion activity is not sedimented by high speed centrifugation and is unaffected by membrane-disrupting treatments. Penicillin N and ACV (presumably via penicillin N) are the only substrates so far accepted by the ring expanding enzyme. New syntheses of penicillin N and isopenicillin N are described.