Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in Streptomyces clavuligerus

Abstract

Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a "supercluster" in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic 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 acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that 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 clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.Streptomyces clavuligerus produces an array of ␤-lactam compounds including cephamycin C, clavulanic acid, and at least four other clavam metabolites. Clavulanic acid has considerable chemotherapeutic and economic value because of its ␤-lactamaseinhibitory activity. In contrast, the other clavam metabolites are ineffective as ␤-lactamase inhibitors. These clavam metabolites have the same nuclear structure, a fused bicyclic ␤-lactam-oxazolidine ring system, as does clavulanic acid. However, the stereochemistry of the ring system is 5R in the clavams rather than 5S as in clavulanic acid, and the clavam metabolites also have different side chain substituents at C-2 (Fig.

Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

“…orf7 (8), located downstream of claR and in the opposite orientation, has 1,668 nucleotides (nt) with a 72% GϩC content and encodes a protein of 555 amino acids. An intergenic region of 164 bp is present between orf6 and orf7 and contains two palindromic sequences (nt 9122 to 9136 and nt 9144 to 9158; nt 9222 to 9228 and nt 9232 to 9238 in U87786) that may form stem-and-loop structures in the RNA with free energies of Ϫ41.8 and Ϫ16.6 kcal/mol.…”

“…This suggests that a gene alternative to ceaS encodes an enzyme able to form the C3 unit from glycerol. Knockout mutants disrupted in bls, pah, or orf6 as well as the above-mentioned ceaS and cas2 were found to be blocked in clavulanic acid and clavam production when grown in the starch-asparagine (SA) medium but partially regained the ability to produce clavulanic acid and clavams when grown in peptone-based soy medium (8). The degree of restoration on soy medium was higher for clavam production, which reached up to 100% of wild-type production for the pah-disrupted strain.…”

“…Additionally it produces compounds with clavam structure, such as the ␤-lactamase inhibitor clavulanic acid or the antibacterial alanylclavam (26), and compounds with pyrrothin structure, such as holomycin (5). Each of these secondary metabolites has different precursors (␣-aminoadipic acid, cysteine, and valine for cephamycin C; arginine for clavulanic acid; and probably cysteine for holomycin), although it is believed that clavulanic acid and clavams share the initial steps of their pathways (8). Genes for clavulanic acid biosynthesis are located adjacent to those of the cephamycin C cluster in S. clavuligerus (7,8,33), forming the so-called ␤-lactam supercluster.…”

“…Each of these secondary metabolites has different precursors (␣-aminoadipic acid, cysteine, and valine for cephamycin C; arginine for clavulanic acid; and probably cysteine for holomycin), although it is believed that clavulanic acid and clavams share the initial steps of their pathways (8). Genes for clavulanic acid biosynthesis are located adjacent to those of the cephamycin C cluster in S. clavuligerus (7,8,33), forming the so-called ␤-lactam supercluster. Several enzymes encoded by these genes have been purified to homogeneity, yielding information on different steps of the pathway (2,9,29,34), and the ␤-lactam synthetase and clavaminate synthetase have been crystallized (17,35).…”

“…Several enzymes encoded by these genes have been purified to homogeneity, yielding information on different steps of the pathway (2,9,29,34), and the ␤-lactam synthetase and clavaminate synthetase have been crystallized (17,35). However, the studies on clavulanic acid biosynthesis are confused by the presence of two sets of paralogous genes that encode the early enzymes of the pathway (8). The cas1 and cas2 genes, which encode the multifunctional clavaminate synthases 1 and 2, are located, respectively, in the clavam gene cluster (18) and in the clavulanic acid gene cluster (15,22).…”