Expansion of the clavulanic acid gene cluster: Identification and in vivo functional analysis of three new genes required for biosynthesis of clavulanic acid by Streptomyces clavuligerus

Clavulanic acid is a potent inhibitor of beta-lactamase enzymes and is of d emonstrated value in the treatment of infections by beta-lactam-resistant b acteria, Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient fo r clavulanic acid biosynthesis, because they allowed production of the anti biotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander , and S. E. Jensen, p. 219-236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identific ation of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are requ ired for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are containe d within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferre doxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to an y known protein. The results of this study extend the biosynthetic gene clu ster for clavulanic acid and attest to the importance of analyzing biosynth etic genes in the context of their natural host. Potential functional roles for these proteins are proposed.