Non-ribosomal peptide synthetases (NRPSs) are responsible for the natural in vivo production of a large number of therapeutically relevant compounds. The non-ribosomal peptides (NRPs) formed by their action are the product of the long modular assembly lines, whilst the terminal step in the biosynthesis is the hydrolytic release, and frequently, macrocyclisation of the aminoacyl-S-thioester by an embedded thioesterase (TE). The surugamide biosynthetic pathway is composed of two NRPS assembly lines. One produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded TE, which led us to question how the peptides are released from the assembly line (and cyclised in the case of surugamide A). We characterised an alpha/beta hydrolase and β-lactamase in vivo and established that the former is a type II TE for surugamide A, but not surugamide F, and that the latter is a trans-acting release factor for both compounds. In vitro substrate utilisation assays unambiguously established that the β-lactamase can produce mature surugamides A and F from N-acetylcysteamine (SNAC) thioester mimics of the cognate terminal biosynthetic intermediates. Using bioinformatics, we estimate that ∼12 % of filamentous Actinobacteria harbor an NRPS system lacking an embedded TE and instead use a trans-acting β-lactamase release strategy. This expands the paradigmatic understanding of how non-ribosomal peptides are released from the terminal NRPS module and adds a new dimension to the synthetic biology toolkit, potentially useful in the search for novel antibiotics.

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