Streptomyces clavuligerus has a second copy of the proclavaminate amidinohydrolase gene

Past genetic studies have indicated that the genes encoding early enzymes of clavulanic acid biosynthesis may be duplicated in Streptomyces clavuligerus. We observed cross-hybridizing bands upon Southern analyses of proclavaminate amidinohydrolase (pah)-defective mutant strains of S. clavuligerus screened with a pah-specific probe. The DNA fragment responsible for this cross hybridization was cloned and sequenced and shown to encode a second copy of the pah gene. The new pah gene (pah1) was 1,056 bp in length, and its sequence was 72% identical to that of the original pah gene (pah2). Disruption mutants with defects in pah1 showed no significant effects on production of clavulanic acid or any of the clavam metabolites with stereochemistries opposite that of clavulanic acid (5S clavams) produced by S. clavuligerus when they were grown on starch asparagine or soy medium. However, double mutants with defects in both pah1 and pah2 were defective in the production of both clavulanic acid and all of the 5S clavam metabolites.