Opening the circle – characterisation of a unique esterase in faulknamycin biosynthesis Asif Fazal 1,2,3 , Ryan F. Seipke 1,3 1 Faculty of Biological Sciences, 2 Faculty of Engineering and Physical Sciences, 3 Astbury Centre for Structural Molecular Biology, University of Leeds, UK Non-ribosomal peptide synthetases (NRPSs) produce a large number of structurally diverse and therapeutically relevant compounds. These long modular assembly lines frequently contain enzymatic domains serving to increase chemical diversity. A further source of enzyme-derived diversity in final chemical scaffolds is through the use of different modes of chain termination, as well as post-assembly line tailoring enzymes. We have previously characterised the standalone peptide cyclase from the surugamide biosynthetic gene cluster (BGC); and found its head-to-tail cyclisation activity is required for compound production. Recently, the discovery of the faulknamycins and its associated BGC added an extra member to the family of cyclic peptides whose NRPS system involves a standalone cyclase enzyme. Interestingly, linear faulknamycins were also observed in culture supernatants and the sequence of these linear compounds was not consistent with NRPS collinearity dogma. The BGC contains multiple genes encoding for putative tailoring enzymes, as well as an esterase enzyme similar to previously characterised erythromycin esterases. We characterised the esterase and cyclase in vivo and showed cyclo-faulknamycin is only formed in the esterase deletion strain, whereas the cyclase was a prerequisite for any faulknamycin production. Complementation of the esterase deletion led to production of only the linear form of the compound, and in vitro assays will be used to further determine the nature of functionality and the purpose of cyclic peptide linearisation. An increased understanding of this class of enzymes could inform the detection, localisation, and characterisation of similar enzymes playing roles of expanded significance within biosynthetic pathways. Standalone enzymes are also of great interest for potential pathway engineering efforts and therefore, insights gained here could aid hugely in attempts to create truly novel “natural” products.
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© The Author(s), 2022
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