A Physically Linked CYP720B–Diterpene Synthase Gene Pair Controls Ferruginol Biosynthesis in Taiwania cryptomerioides and Reveals Conserved Genome Organization in Cupressidae

Members of the Cupressaceae produce abundant phenolic abietane-type diterpenoids such as ferruginol, a compound that is thought to contribute to heartwood durability and to serve as the precursor to medically relevant taiwaniaquinoids in Taiwania cryptomerioides . However, the oxidative steps downstream of the diterpene precursor levopimaradiene remain poorly understood. Here, we identified a physically linked cytochrome P450 ( CYP720B )– diterpene synthase ( DTS ) gene pair in the Cryptomeria japonica genome and cloned the corresponding wood-expressing homologs from T . cryptomerioides . Functional expression in Saccharomyces cerevisiae and Escherichia coli demonstrated that two CYP720Bs catalyze oxidative conversion of levopimaradiene, while the adjacent, newly isolated DTS (TcKSL9) functions as a putative levopimaradiene synthase. Phylogenetic analyses revealed that Cupressaceae CYP720Bs form a lineage distinct from Pinaceae homologs. Guided by this relationship, comparative genomic surveys identified head-to-head CYP720B – DTS gene pairs across multiple Cupressidae genomes. Subsequent phylogenetic analyses of paired genes, together with microsynteny comparisons, revealed conserved local genomic neighborhoods and reciprocal best-hit relationships among Cupressidae species, patterns consistent with an early establishment and evolutionary persistence of this genomic configuration within the clade. Collectively, our findings elucidate the enzymatic steps leading to ferruginol formation in T. cryptomerioides and reveal a conserved CYP720B–DTS genomic association within Cupressidae, providing new insight into diterpenoid biosynthesis and genome organization in conifers. • CYP720B-DTS gene pairs are widely distributed in Cupressidae genome. • Taiwania homologs form a functional pathway producing ferruginol. • Cupressidae gene pairs likely originated from a shared ancestral event.