Characterization of Three Tailoring Enzymes in Dutomycin Biosynthesis and Generation of a Potent Antibacterial Analogue
2016-05-11T00:00:00Z (GMT) by
The anthracycline natural product dutomycin and its precursor POK-MD1 were isolated from Streptomyces minoensis NRRL B-5482. The dutomycin biosynthetic gene cluster was identified by genome sequencing and disruption of the ketosynthase gene. Two polyketide synthase (PKS) systems are present in the gene cluster, including a type II PKS and a rare highly reducing iterative type I PKS. The type I PKS DutG repeatedly uses its active sites to create a nine-carbon triketide chain that is subsequently transferred to the α-l-axenose moiety of POK-MD1 at 4″-OH to yield dutomycin. Using a heterologous recombination approach, we disrupted a putative methyltransferase gene (dutMT1) and two glycosyltransferase genes (dutGT1 and dutGT2). Analysis of the metabolites of these mutants revealed the functions of these genes and yielded three dutomycin analogues SW140, SW91, and SW75. The major product SW91 in Streptomyces minoensis NRRL B-5482-ΔDutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the in vitro enzymatic assay. DutGT1 and DutGT2 were found to be responsible for the introduction of β-d-amicetose and α-l-axenose, respectively. Dutomycin and SW91 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus, whereas POK-MD1 and SW75 had no obvious inhibition, which revealed the essential role of the C-4″ triketide chain in antibacterial activity. The minimal inhibitory concentration of SW91 against the two strains was 0.125 μg mL–1, lower than that of dutomycin (0.25 μg mL–1), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification.
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