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. 2008 Dec;190(24):7957-65.
doi: 10.1128/JB.00698-08. Epub 2008 Oct 17.

Alanylclavam biosynthetic genes are clustered together with one group of clavulanic acid biosynthetic genes in Streptomyces clavuligerus

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Alanylclavam biosynthetic genes are clustered together with one group of clavulanic acid biosynthetic genes in Streptomyces clavuligerus

Nathan J Zelyas et al. J Bacteriol. 2008 Dec.

Abstract

Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway.

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Figures

FIG. 1.
FIG. 1.
Proposed biosynthetic pathway leading to clavulanic acid and the 5S clavams in S. clavuligerus.
FIG. 2.
FIG. 2.
The paralogue gene cluster. Gray arrows represent ORFs of the previously described paralogue gene cluster. Black arrows represent newly discovered ORFs specifically required for alanylclavam biosynthesis. White arrows indicate newly discovered ORFs not required for alanylclavam biosynthesis.
FIG. 3.
FIG. 3.
HPLC analysis of clavam metabolites produced by wild-type and mutant strains of S. clavuligerus. Wild type (A), orfA mutant (B), orfB mutant (C), orfC mutant (D), and orfD mutant (E) are shown. AC, alanylclavam; C2C, clavam-2-caboxylate; HMC, 2-hydroxymethylclavam; CA, clavulanic acid; NC, novel clavam. The faint dotted line shows the expected location of the novel clavam in the HPLC profile from the wild-type strain.
FIG. 4.
FIG. 4.
MS analysis of the novel clavam accumulated by orfC and orfD mutants of S. clavuligerus. HPLC profile of orfD mutant of S. clavuligerus (A), mass spectrum of the novel clavam peak (retention time, 3.62 min) seen in panel A (B), HPLC profile of wild-type S. clavuligerus (C), and mass spectrum of the alanylclavam peak (retention time, 3.77 min) seen in panel C (D) are shown. Abbreviations for metabolite peaks are given in legend to Fig. 3.
FIG. 5.
FIG. 5.
HPLC analysis of clavam metabolites produced by the complemented orfC mutant of S. clavuligerus. Wild type (A), orfC mutant (B), complemented orfC mutant (C), and overlay and expansion of panels A to C in the region of retention times from 2.8 to 3.5 min (D) are shown. The faint dotted line indicates the presence of low levels of alanylclavam in the complemented orfC mutant. Abbreviations for metabolite peaks are given in legend to Fig. 3.

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