Genome Mining and Genetic Manipulation Reveal New Isofuranonaphthoquinones in Nocardia Species

The identification of specialized metabolites isolated from microorganisms is urgently needed to determine their roles in treating cancer and controlling multidrug-resistant pathogens. Naphthoquinones act as anticancer agents in various types of cancers, but some toxicity indicators have been limited in their appropriate application. In this context, new isofuranonaphthoquinones (ifnq) that are less toxic to humans could be promising lead compounds for developing anticancer drugs. The aim of this study is to identify and characterize novel furanonaphthoquinones (fnqs) from sp. CS682 and to evaluate their potential therapeutic applications. Analysis of the genome of sp. CS682 revealed the presence of a furanonaphthoquinone ( ) gene cluster, which displays a similar genetic organization and high nucleotide sequence identity to the gene cluster from sp. RI-77, a producer of the naphthoquinones JBIR-76 and JBIR-77. In this study, the overexpression of the antibiotic regulatory protein (SARP) in sp. CS682DR (nargenicin gene-deleted mutant) explicitly produced new fnqs, namely, NOC-IBR1 and NOC-IBR2. Subsequently, the role of the SARP regulator was confirmed by gene inactivation using CRISPR-Cas9 and complementation studies. Furthermore, antioxidant, antimicrobial, and cytotoxicity assays were performed for the isolated compounds, and it was found that NOC-IBR2 exhibited superior activities to NOC-IBR1. In addition, a flexible methyltransferase substrate, ThnM3, was found to be involved in terminal methylation of NOC-IBR1, which was confirmed by in vitro enzyme assays. Thus, this study supports the importance of genome mining and genome editing approaches for exploring new specialized metabolites in a rare actinomycete called .