In vitro biosynthesis of the antitumor agent azinomycin B.
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[reaction: see text] Azinomycins have potential therapeutic value as antitumor agents; however, their biosynthesis is poorly understood. Here, we provide the first demonstration of a protein cell-free system capable of supporting complete in vitro biosynthesis of the antitumor agent azinomycin B. The cell-free system is utilized to probe the cofactor dependence and substrate requirements of the pathway en route to azinomycin.