Expansion of the domain-spanning borosin family of RiPP natural products Aileen Lee 1 , Aman S. Imani 1 , Nisha Vishwanathan 2 , Yudi Rusman 3 , Christine Salomon 3 , Michael F. Freeman 1,2 1 University of Minnesota, Department of Biochemistry, Molecular Biology, & Biophysics, USA, 2 University of Minnesota, Biotechnology Institute, USA, 3 University of Minnesota, Center for Drug Design, USA One of the more challenging hurdles that has plagued the natural products field is the high rediscovery rate of small molecules that have already been identified. This problem highlights the need to look in areas which have traditionally been less explored. Fungi have long been recognized as prolific producers of natural products 1. Despite this, the vast landscape of fungal natural products remains largely underexplored due in large part to the difficulty in finding suitable cultivation conditions as well as a lack of genetic tools for these organisms. However, recent technological advances have made pursuit of these small molecules much more accessible. In this study, we expand on our work with the borosin family of ribosomally synthesized and post-translationally modified peptides (RiPPs). Borosins were originally discovered in fungi and are characterized by the presence of backbone α- N -methylations which were once believed to exclusively be produced non-ribosomally 2,3. These modifications have garnered some interest as α- N -methylated peptides have been shown to confer favorable pharmacokinetic properties including increased stability and bioavailability 4. Here, we used methods in genomics, molecular biology, and analytical chemistry to show that a previously isolated set of potently antiproliferative cyclic α- N - methylated peptides—the gymnopeptides, isolated from the unsequenced mushroom Gymnopus fusipes —are borosins 5,6. Furthermore, we identified several previously uncharacterized gymnopeptides that are produced by the fungus. Additionally, after observing similar BGCs in bacteria, we leveraged our knowledge of fungal borosins to explore the prevalence of these systems in available genomes of non-fungal organisms using bioinformatic tools. This revealed a group of putative borosin BGCs displaying highly diverse protein architectures in the core biosynthetic proteins 7 . This work highlights our efforts in pursuing natural products from both a traditionally underexplored group of organisms as well as from the unknown sequence space of already available genomes. References 1. MT Robey, LK Caesar, MT Drott, and NL Kelleher. (2021). PNAS, 118(19) 2. N van der Velden, N Kälin, MJ Helf, J Piel, MF Freeman, and MKünzler. (2017). Nat. Chem. Biol. ,13: 821-822 3. S Ramm, B Krawczyk, A Mühlenweg, A Poch, E Mösker, and RDSüssmuth. (2017). Angew. Chem. Int. Ed. Engl ., 56: 9994- 9997 4. J Chatterjee, F Rechenmacher, and H Kessler. (2013). Angew. Chem. Int. Ed. , 52:254-269 5. AVanyolos et al. (2016). Org. Lett ., 18: 2688-2691 6. MR Quijano, C Zach, FS Miller, AR Lee, AS Imani,MKünzler, and MF Freeman. (2019). J. Am. Chem. Soc ., 141(24): 9637- 9644 7. AS Imani, AR Lee, N Vishwanathan, F deWaal, and MF Freeman. (2022). ACS Chem. Biol.
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