Thioesterase from Cereulide Biosynthesis Is Responsible for Oligomerization and Macrocyclization of a Linear Tetradepsipeptide

Cereulide 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.