Five Additional Genes Are Involved in Clavulanic Acid Biosynthesis in Streptomyces clavuligerus

Abstract

An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for ␤-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

A Two-component Regulatory System Involved in Clavulanic Acid Production

“…It has been previously reported that a series of partial structural genes are involved in the CA biosynthetic pathway [24]. While these genes accounted for many of the expected activities, it was not clear that all of the genetic information needed to form CA was present in this region.…”

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

“…The assay was initiated with 1 mM potential carboxylate substrates in a final volume of 1 ml. (32), respectively. The two proposed versions of orf17 were cloned into the pET24a(ϩ) vector.…”

“…C 18 octadecylsilyl guard cartridges (SecurityGuard, Phenomenex) were used throughout and replaced as required. The method was modified from Jensen et al (32). Samples were eluted with a gradient from 5 to 30% acetonitrile in 100 mM ammonium formate, pH 4.0, at a flow rate of 1 ml⅐min Ϫ1 over 15 min followed by a wash of 30% acetonitrile in 100 mM ammonium formate, pH 4.0, for 10 min before the column was re-equilibrated to 5% acetonitrile in 100 mM ammonium formate, pH 4.0, over 5 min.…”

“…1c), N-acetyl-cla-* This work was supported by grants from the Biotechnology and Biological Sciences vaminic acid, N-acetyl-glycyl-clavaminic acid, and N-glycyl-clavaminic acid have been isolated from a mutant strain of S. clavuligerus deficient in clavulanic acid production (designated dcl8) (33). Recently, the latter two of these compounds have been reported in disruption mutants of orf15 and orf16 (32). These compounds have not been observed in disruption mutants of any of the other orfs of the clavulanic acid biosyn- (4 -7).…”

“…b, identified steps in the clavulanic acid biosynthetic pathway. c, structures of the accumulated products from the dcl8, orf15, and orf16 disruption mutants (32,33). d, outline mechanism for a generic carboxylate-amine ligation reaction catalyzed by an ATP-grasp enzyme.…”