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 <i>Streptomyces minoensis</i> 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 (<i>dutMT1</i>) and two glycosyltransferase genes (<i>dutGT1</i> and <i>dutGT2</i>). 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 <i>Streptomyces minoensis</i> NRRL B-5482-ΔDutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the <i>in vitro</i> 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 <i>Staphylococcus aureus</i> and methicillin-resistant <i>S. aureus</i>, 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<sup>–1</sup>, lower than that of dutomycin (0.25 μg mL<sup>–1</sup>), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification.
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