Cloning, sequencing and disruption of a gene from Streptomyces clavuligerus involved in clavulanic acid biosynthesis

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis

“…Serendipitously, this gene lay directly upstream of cs2 (23) and in a region later reported to be contained within the clavulanic acid gene cluster (45). Importantly, insertional inactivation of this gene dramatically reduced clavulanic acid levels but left cephamycin production essentially unchanged (1). In recognition of its clear relation to clavulanic acid biosynthesis, acvsr was renamed cla (1); its reported sequence is identical to that determined to be pah in the course of the present work.…”

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

“…5). To obtain a larger DNA fragment downstream of orf9 (cad), a genomic sublibrary was constructed based on the restriction map described by Aidoo et al (1). Genomic DNA isolated from S. clavuligerus was digested with EcoRI-KpnI, and the fragments between 8.5 and 10 kb were isolated and ligated into pBluescript II SK(Ϫ).…”

“…The gene products of orf4 and orf5, proclavaminate amidino hydrolase (PAH) and clavaminate synthase isozyme 2 (CS2), respectively, function alternately to carry deoxyguanidinoproclavaminic acid by hydroxylation to guanidinoproclavaminic acid (12), to mediate an arginase-like reaction to proclavaminic acid (Fig. 1, compound 4) (1,65), and to govern successive oxidative cyclization and desaturation to clavaminic acid ( Fig. 1, compound 5) (13,18,23,54).…”

ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

“…␤-Lactam formation is catalyzed by ␤-lactam synthetase (BLS) (15,16), to give deoxyguanidinoproclavaminic acid (17), which is then converted in four steps to (3S,5S)-clavaminic acid. Three of these steps are catalyzed by a single 2-oxoglutarate (2-OG)-dependent oxygenase, clavaminic acid synthase (CAS2) (10, 18 -20), and one is catalyzed by proclavaminate amidinohydrolase (PAH) (21), to give the bicyclic clavam ring system (22)(23)(24). Crystal structures have been reported for the first four enzymes in the pathway (25)(26)(27)(28) as well as for the gene product of orf6 (OAT2), an ornithine acetyltransferase (29,30) proposed to be involved in the biosynthesis of the L-arginine feedstock for the pathway.…”