Enzymes catalyzing the early steps of clavulanic acid biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus

Genes encoding the proteins required for clavulanic acid biosynthesis and f or cephamycin biosynthesis are grouped into a "supercluster" in Streptomyce s clavuligerus. Nine open reading frames (ORFs) associated,vith clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, incl uding six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavul anic acid biosynthesis is unclear, and one ORF not previously reported. Evi dence for the involvement of these ORFs in clavulanic acid production was o btained by generating mutants and showing that all were defective for clavu lanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic aci d-producing ability was restored. This ability to produce clavulanic acid w hen seemingly essential biosynthetic enzymes have been mutated suggests tha t paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy- based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavul anic acid and the other clavam metabolites produced by this organism, No ev idence was seen for paralogues of the four remaining genes involved in late , clavulanic acid-specific steps in the pathway.