Thermorubin Biosynthesis Initiated by a Salicylate Synthase Suggests an Unusual Conversion of Phenols to Pyrones

Thermorubin is a tetracyclic naphthoisocoumarin natural product that demands investigation due to its novel mechanism of bacterial protein synthesis inhibition and its unusual structural features. In this work, we describe the identification of the biosynthetic cluster responsible for thermorubin from the sequenced Laceyella sacchari producer species and its confirmation via heterologous production in Escherichia coli. Based on an in-depth annotation of the cluster, we propose a biosynthetic pathway that accounts for the formation of the unique, nonterminal pyrone. Additionally, the expression and use of salicylate synthase TheO enabled testing of the stability properties of this extremophile-derived enzyme. TheO displayed rapid kinetics and a remarkably robust secondary structure, converting chorismate to salicylate with a K_M of 109 ± 12 μM, k_cat of 9.17 ± 0.36 min^-1, and catalytic efficiency (k_cat/K_M) of 84 ± 9 nM^-1 min^-1, and retained significant activity up to 50 °C. These studies serve as the basis for continued biosynthetic investigations and bioinspired synthetic approaches.