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Characterization of Three Tailoring Enzymes in Dutomycin Biosynthesis and Generation of a Potent Antibacterial Analogue
journal contribution
posted on 2016-05-11, 00:00 authored by Lei Sun, Siyuan Wang, Shuwei Zhang, Lei Shao, Qian Zhang, Chad Skidmore, Cheng-Wei
Tom Chang, Dayu Yu, Jixun ZhanThe 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.