Abstract

Natural products (NPs) are synthesized by biosynthetic gene clusters (BGCs), whose genes are involved in producing one or a family of chemically related metabolites. Advances in comparative genomics have been favourable for exploiting huge amounts of data and discovering previously unknown BGCs. Nonetheless, studying distribution patterns of novel BGCs and elucidat-ing the biosynthesis of orphan metabolites remains a challenge. To fill this knowledge gap, our study developed a pipeline for high-quality comparative genomics for the actinomycete genus Rhodococcus, which is metabolically versatile, yet under-studied in terms of NPs, leading to a total of 110 genomes, 1891 BGCs and 717 non-ribosomal peptide synthetases (NRPSs). Phylogenomic inferences showed four major clades retrieved from strains of several ecological habitats. BiG- SCAPE sequence similarity BGC networking revealed 44 unidentified gene cluster families (GCFs) for NRPS, which presented a phylogenomic-dependent evolution pattern, supporting the hypothesis of vertical gene transfer. As a proof of concept, we analysed in- depth one of our marine strains, Rhodococcus sp. H- CA8f, which revealed a unique BGC distribution within its phylogenomic clade, involved in producing a chloramphenicol-related compound. While this BGC is part of the most abundant and widely distributed NRPS GCF, CORASON analysis unveiled major differences regarding its genetic context, co-occurrence patterns and modularity. This BGC is composed of three sections, two well- conserved right/left arms flanking a very variable middle section, composed of nrps genes. The presence of two non-canonical domains in H-CA8f's BGC may contribute to adding chemical diversity to this family of NPs. Liquid chromatography- high resolution MS and dereplication efforts retrieved a set of related orphan metabo-lites, the corynecins, which to our knowledge are reported here for the first time in Rhodococcus. Overall, our data provide insights to connect BGC uniqueness with orphan metabolites, by revealing key comparative genomic features supported by models of BGC distribution along phylogeny.