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In Streptomyces clavuligerus, the gene cluster involved in the biosynthesis of the clinically used β-lactamase inhibitor clavulanic acid contains a gene (orf12 or cpe) encoding a protein with a C-terminal class A β-lactamase-like domain. The cpe gene is essential for clavulanic acid production, and the recent crystal structure of its product (Cpe) was shown to also contain an N-terminal isomerase/cyclase-like domain, but the function of the protein remains unknown. In the current study, we show that Cpe is a cytoplasmic protein and that both its N- and C-terminal domains are required for in vivo clavulanic acid production in S. clavuligerus. Our results along with those from previous studies allude towards a biosynthetic role for Cpe during the later stages of clavulanic acid production in S. clavuligerus. Amino acids from Cpe essential for biosynthesis were also identified, including one (Lys89) from the recently described N-terminal isomerase-like domain of unknown function. Homologues of Cpe from other clavulanic acid-producing Streptomyces spp. were shown to be functionally equivalent to the S. clavuligerus protein, whereas those from non-producers containing clavulanic acid-like gene clusters were not. The suggested in vivo involvement of an isomerase-like domain recruited by an ancestral β-lactamase related protein, supports a previous hypothesis that Cpe could be involved in a step requiring the opening and modification of the clavulanic acid core during its biosynthesis from 5S precursors., Competing Interests: The authors have declared that no competing interests exist.

Clavulanic acid is a bacterial specialized metabolite, which inhibits certain serine β-lactamases, enzymes that inactivate β-lactam antibiotics to confer resistance. Due to this activity, clavulanic acid is widely used in combination with penicillin and cephalosporin (β-lactam) antibiotics to treat infections caused by β-lactamase-producing bacteria. Clavulanic acid is industrially produced by fermenting Streptomyces clavuligerus , as large-scale chemical synthesis is not commercially feasible. Other than S. clavuligerus , Streptomyces jumonjinensis and Streptomyces katsurahamanus also produce clavulanic acid along with cephamycin C, but information regarding their genome sequences is not available. In addition, the Streptomyces contain many biosynthetic gene clusters thought to be "cryptic," as the specialized metabolites produced by them are not known. Therefore, we sequenced the genomes of S. jumonjinensis and S. katsurahamanus , and examined their metabolomes using untargeted mass spectrometry along with S. clavuligerus for comparison. We analyzed the biosynthetic gene cluster content of the three species to correlate their biosynthetic capacities, by matching them with the specialized metabolites detected in the current study. It was recently reported that S. clavuligerus can produce the plant-associated metabolite naringenin, and we describe more examples of such specialized metabolites in extracts from the three Streptomyces species. Detailed comparisons of the biosynthetic gene clusters involved in clavulanic acid (and cephamycin C) production were also performed, and based on our analyses, we propose the core set of genes responsible for producing this medicinally important metabolite. [ABSTRACT FROM AUTHOR]