12The terminal step in the biosynthesis of non-ribosomal peptides is the hydrolytic release, and 13 frequently, macrocyclization of an aminoacyl-S-thioester by an embedded thioesterase. The 14 surugamide biosynthetic pathway is composed of two NRPS assembly lines where one produces 15 surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear 16 decapeptide. The terminal module of each system lacks an embedded thioesterase, which led us to 17 question how the peptides are released from the assembly line (and cyclized in the case of surugamide 18 A). We characterized a cyclase belonging to the -lactamase superfamily (SurE) in vivo and 19 established that it is a trans-acting release factor for both compounds and verified this functionality 20 in vitro with a thioester mimic of linear surugamide A. Using bioinformatics, we estimate that ~11% 21 of filamentous Actinobacteria harbor an NRPS system lacking an embedded thioesterase and instead 22 employ a trans-acting cyclase. This study expands the paradigmatic understanding of how non-23 ribosomal peptides are released from the terminal PCP and adds a new dimension to the synthetic 24 biology toolkit. 25 Non-ribosomal peptides (NRPs) are a large family of structurally complex and diverse natural 26 products, often with biologically and therapeutically relevant activities. They are synthesized by large 27 multifunctional enzymes called non-ribosomal peptide synthetases (NRPSs), which are organised 28 into relatively independently functioning modules that work in an assembly line-like manner until the 29 final polypeptide structure is generated. 1 During biosynthesis, the growing peptide chain remains 30 covalently linked to the 4´-phosphopantetheinyl cofactor of the peptidyl carrier protein (PCP) 31 domains. The terminal module usually possesses a C-terminal thioesterase (TE) domain, which off-32 loads the polypeptide intermediate from the PCP on to a conserved serine residue whereby either a 33 hydrolytic or macrocylization reaction occurs to produce the mature peptide. 2 34 Surugamide A (1) and associated minor products B-E (2-5) are cyclic octapeptides initially 35 identified from Streptomyces sp. JAMM992 as inhibitors of cathepsin B, which also possess 36 antibacterial activity. 3,4 Identification of the surugamide (sur) biosynthetic gene cluster (BGC) from 37this organism revealed the presence of four NRPS genes (surABCD), which encoded 18 biosynthetic 38 modules (Figure 1, Scheme 1). The eight modules encoded by SurAD were consistent with the 39 biosynthesis of 1-5, whereas the remaining 10 modules encoded by SurBC were shown to direct the 40 biosynthesis of an unrelated linear decapeptide named, surugamide F (7), which is structurally similar 41 to gramicidin A. 5 S. albus J1074 was recently shown to produce surugamides, including an unusual 42 derivative named acyl-surugamide A (6), which has antifungal bioactivity. 6,7 We identified the same 43 BGC in a related strain, S. albus S4 8 and verified that it produced the major produ...

SignificanceHistidine biosynthesis is a target for herbicide and antibacterial agents, with imidazoleglycerol-phosphate dehydratase (IGPD) a key enzyme within this pathway. As a result, IGPD is the focus of inhibitor design programs, with several potent herbicides in development. Interestingly, the lead inhibitor is more potent against yeast (Saccharomyces) compared with plant (Arabidopsis) IGPD. To understand this change, we have determined their structure by electron microscopy to reveal a possible mechanism behind differences in inhibitor potency, with Saccharomyces IGPD containing a 24-amino acid insert that forms an extended surface loop that stabilizes an inhibitor binding loop. This study provides insights into the IGPD family and demonstrates the power of using an electron microcopy approach to study inhibitor binding.

Microbial natural products underpin the majority of antimicrobial compounds in clinical use and the discovery of new effective antibacterial treatments is urgently required to combat growing antimicrobial resistance. Non-ribosomal peptides are a major class of natural products to which many notable antibiotics belong. Recently, a new family of non-ribosomal peptide antibiotics were discovered—the desotamide family. The desotamide family consists of desotamide, wollamide, surugamide, ulleungmycin and noursamycin/curacomycin, which are cyclic peptides ranging in size between six and ten amino acids in length. Their biosynthesis has attracted significant attention because their highly functionalised scaffolds are cyclised by a recently identified standalone cyclase. Here, we provide a concise review of the desotamide family of antibiotics with an emphasis on their biosynthesis.

Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign biosynthetic gene clusters by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism. Keywords Streptomyces Á Streptomyces albus Á Secondary metabolites Á Natural products Á Heterologous expression Asif Fazal and Divya Thankachan have contributed equally to this work.