Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which e ncodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and se quenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading fra me located immediately upstream of cas1, were constructed by a gene replace ment procedure. Similar techniques were used to generate S. clavuligerus mu tants carrying a deletion that encompassed portions of the two open reading frames, cvm1 and cvm5, located directly downstream of cas1. Both classes o f mutants still produced clavulanic acid and cephamycin C but lost the abil ity to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2- hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosy nthesis. When a cas1 mutant of S. clavuligerus was constructed by gene repl acement, it produced lower levels of both clavulanic acid and most of the a ntipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic ac id nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common poo l of clavaminic acid that is shunted toward clavulanic acid and clavam meta bolite biosynthesis.