Identification of Cembrene Synthases Uncovers Polyphyletic Origins of 14-Membered Carbocyclic Cembranoids Biosynthesis in Corals

Abstract

Genome mining is an efficient strategy for natural product discovery, yet its application to terpene synthases frequently results in the repeated identification of identical products. Cembranoids, a class of coral-derived natural products featuring 14-membered carbocyclic scaffolds, present formidable challenges for chemical synthesis. To date, only two biosynthetic precursors have been synthesized across fifteen coral enzymes from six independent studies. To address these limitations, we developed Ariadne, an integrated platform for genome-wide targeted mining of terpene synthases from corals. Using this platform, we identified five cembrene synthases, including two with high sequence similarity to known cembrene B synthases and three low-similarity enzymes experimentally validated through heterologous expression in yeast, achieving a prediction accuracy of 80% from ninety terpene synthase homologs. Leveraging the rapid identification of novel cembrene synthases, phylogenetic analysis further uncovered the evolutionary trajectory of this enzyme family from coral, providing valuable guidance for the engineering of an early-diverging enzyme, which is capable of synthesizing an unreported cembrene scaffold by the coral enzyme. Collectively, this work establishes a new paradigm for terpene synthase discovery and substantially advances the genome mining in marine animals as well as the combinatorial biosynthesis of marine-derived natural products.