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.

PURIFICATION AND MOLECULAR WEIGHT DETERMINATION OF KERATINASE ISOLATED FROM STREPTOMYCES MALAYSIENSIS

Objective: The aim of the present study was to purify and determine the molecular weight of keratinase isolated from Streptomyces malaysiensis.Methods: For that purpose purification was done using ammonium sulphate and Sephadex-LH 100 column chromatography. Further, the fractions were pooled and subjected to molecular weight determination using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).Results: The obtained results showed keratinase with 47.57% recovery, 3.5-fold purification and an estimated molecular mass of 27,000 Da. Keratinase showed an optimal activity at 60 οC and pH 8. Keratinase activity of the purified product was assayed with feather powder as a substrate. The isolated strain was identified as Streptomyces malaysiensis based on phylogenetic tree analysis. The strain isolated from termite mound soil showed the highest keratinase activity, which could be considered a microorganism of environmental origin.Conclusion: The production of keratinase on simple media with feathers as sole source allowing its production from the cheap substrate and a commercial production with low production cost. Stability in the presence of detergents, surfactants and solvents make this keratinase extremely useful for a biotechnological process involving keratin.

Isoafricanol synthase from Streptomyces malaysiensis

A terpene cyclases from Streptomyces malaysiensis was characterised as (+)-isoafricanol synthase and its mechanism was investigated using isotopically labelled substrates.

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.