The Lysobacter lactamgenus YK90 pcbAB gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is located immediately upstream of the pcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. The pcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within the pcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. the domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthetase 2 of Bacillus brevis. The pcbAB gene was expressed under the control of the lac promoter in Pseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis in P. putida led to the accumulation of beta-lactam antibiotics. Deletion analysis of an open-reading frame located between the cefE and cefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis in L. lactamgenus appear to be clustered in the order pcbAB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.