Identification of a novel cacaoidin derivative obtained by the heterologous expression of the cao biosynthetic gene cluster Fernando Román-Hurtado, Sánchez-Hidalgo, Marina; Martín, Jesús; Ortiz-López, Francisco Javier; Genilloud, Olga Fundación MEDINA, Spain The increasing emergence of antibiotic resistant pathogenic strains has renewed the interest to discover novel antibiotics, taking advantage of state-of-the-art analytic, metabolomic and genomic tools 1 . Historically, actinomycetes, especially the genus Streptomyces , have been one of the most prolific sources of novel antibiotics 2 . The strain Streptomyces cacaoi CA-170360, from Fundación MEDINA microbial collection, produces cacaoidin, the first member of the new class V lanthipeptides RiPP family 3 , with bioactivity against Gram-positive pathogens, such as Methicillin-Resistant Staphylococcus aureus (MRSA) and Clostridium difficile . The molecule presents unprecedented structural features: an unusually high number of D-amino acids, an N,N -dimethyl lanthionine and an O-glycosylated tyrosine carrying a non-previously reported disaccharide formed by α-L-rhamnose and β-L-6-deoxygulose 3 . The genome of this strain was sequenced and analyzed, and the biosynthetic gene cluster encoding cacaoidin was identified and successfully expressed in the heterologous host S. albus J1074 4 . In this work, we describe the identification of a new variant of cacaoidin in the fermentations of the heterologous host which harbors a different attached disaccharide. We also describe the generation of knockouts that allowed to assign the functions of several proteins of the cluster: Cao4 is involved in the N,N -dimethylation of the N-terminal Ala residue and the glycosyltransferases Cao8 and Cao16 work cooperatively to O-glycosylate the tyrosine residue. References 1. Miethke et al. (2021). Nat. Rev. Chem. 5, 726–749.De Simeis et al. (2021). Antibiotics 10, 483. 2. Ortiz-López et al. (2020). Angew. Chem. Int. Ed. 59, 12654-12658. 3. Román-Hurtado et al. (2021). Antibiotics (Basel) . 10, 403.
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