Clethramycin

Three polyene antibiotics, mediomycins A (1), B (2), and clethramycin (3), were isolated from Streptomyces mediocidicus ATCC23936. Their structures were elucidated through extensive NMR study coupled with chemical reactions and MS/MS fragmentation analysis. All three compounds are linear polyenes consisting of a conjugated oxo-triene group and a hexaene moiety. Compounds 1 and 2 are new polyenes. All three compounds demonstrated a broad spectrum of antifungal activity in vitro.

Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order.

Clethramycin (1) and mediomycin A (2) belong to the linear polyene polyketide (LPP) family of antibiotics that exhibit potent antifungal activity. Structural similarities exist between 1 and 2, except that 2 contains an amino moiety substituted for the guanidino moiety. Herein, the draft genome sequence of Streptomyces mediocidicus ATCC23936, a strain which produces both 1 and 2, was obtained through de novo sequencing. Bioinformatic analysis of the genome revealed a clethramycin (cle) gene cluster that contained 25 open reading frames (orfs). However, amidinohydrolase for 2 formation was not found in the cle gene cluster. Further genomic analysis revealed an amidinohydrolase MedX, which can hydrolyse the guanidino form (1) into the amino form (2) via heterologous co-expression of the cle cluster in Streptomyces lividans or by in vitro catalysis. These results also suggest the feasibility of engineering novel LPPs for drug discovery by manipulating the biosynthetic machinery of S. mediocidicus.