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is a cyclic depsidodecapeptide produced in Bacillus
cereus by two non-ribosomal peptide synthetases, CesA and CesB. While
highly similar in structure and with a homologous biosynthetic gene cluster to
valinomycin, recent work suggests that cereulide is produced via a different
mechanism, which relys on a non-canonical coupling of two didepsipeptide-PCP
bound intermediates. Ultimately this alternative
mechanism generates a tetradepsipeptide-PCP bound intermediate that is prosed
to differ from the tetradepsipeptide predicted from canonical activity of CesA
and CesB. To test this hypothesis, we
chemically synthetize both tetradepsipeptides as N-acetyl cysteamine thioesters
and probed the ability of the purified recombinant terminal CesB thioesterase (CesB
TE) to oligomerize and macrocyclize each substrate. Only the canonical substrate is converted cereulide,
ruling out this alternative mechanism. We also show that CesB TE can use related tertradepsipeptide
substrates, such as the valinomycin tetradespipetide and a hybride
cereulide-valinomycin tetradespispetide in conjunction with its native
substrate to generate chimeric natural products. This work clarifies the biosynthetic origins
of cereulide and provides a powerful biocatalyst to access analogs of these ionophoric
forming natural products.