Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes.

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.

Characterisation of a Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes.

The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential.