Genome Mining of the Strain Streptomyces sp. CA-170360: Identification and Heterologous Expression of Secondary Metabolite Biosynthetic Gene Clusters

Zusammenfassung

The emergence of antibiotic resistant pathogenic strains, especially Gram-negative bacteria, has seriously increased in the last decades, endangering the efficiency of the antibiotics used thus far. As a result, the discovery of new molecules with potential antimicrobial activity has become an essential matter. Historically, actinomycetes, particularly species from the genus Streptomyces, have been one of the most prolific sources of novel antibiotics. The strain Streptomyces cacaoi CA-170360 from the microbial collection of Fundación MEDINA produces the novel cyclic pentapeptides pentaminomycins A-H, alongside the already known cyclic pentapeptides BE-18257 antibiotics, with a moderate antibacterial activity against Acinetobacter baumannii. This strain also produces cacaoidin, the first member of the new class V lanthipeptides (or lanthidins) RiPP family, with bioactivity against Gram-positive pathogens, such as Methicillin-Resistant Staphylococcus aureus (MRSA) and Clostridium difficile. The genome of this strain was sequenced and analyzed, and the biosynthetic gene clusters responsible of the production of these compounds were identified, together with other biosynthetic gene clusters involved in the production of different known secondary metabolites detected in the fermentations of this strain. Pentaminomycins A-H and BE-18257 antibiotics are two different families of cyclopeptides synthesized by two independent non-ribosomal peptide synthetases encoded in tandem within the same biosynthetic gene cluster (cpp). The cluster lacks a thiosterase domain to release and cyclize the pentapeptides; however, it contains a stand-alone PBP-type protein with a betalactamase conserved domain (CppA) upstream the first NRPS gene. The heterologous expression of the cpp cluster in the host Streptomyces albidoflavus J1074 confirmed its implication in the biosynthesis of both pentaminomycins and BE-18257 antibiotics, while the generation of a knockout by genetic replacement of cppA demonstrated the involvement of this protein in the release and cyclization of the peptide chains of both cyclopeptide families. Cacaoidin is a novel glycosylated lanthipeptide with remarkable unprecedented structural features, such as an unusually high number of D-amino acids, an N,N-dimethyl lanthionine system (NMe2Lan), not previously found in known lanthipeptides, and an O-glycosylated Tyr residue with a not previously reported disaccharide formed by α-L-rhamnose and β-L-6-deoxygulose. The available predictive tools were not able to detect the biosynthetic gene cluster responsible of the production of cacaoidin, which was found using the C-terminal amino acid sequence of the structural peptide. The final cao cluster was determided after BLAST analysis and HHpred secondary structure prediction. The heterologous expression of the cluster in S. albidoflavus J1074 confirmed the involvement of the cluster in the biosynthesis of cacaoidin and led to the identification of a new variant of cacaoidin with a different disaccharide lacking two oxygen atoms (cacaoidin-2O). The generation of knockout strains in the heterologous host by genetic replacement allowed the assignment of different roles in several genes in the biosynthesis of cacaoidin: cao4 is involved in the N,N-dimethylation of the N-terminal Ala residue and cao8 and cao16 are two glycosyltransferases working cooperatively in the Tyr O-glycosylation. The generation of the aglycon of cacaoidin was achieved by the controlled acid hydrolysis of pure cacaoidin-2O.