Lineage-specific enzyme optimization underlies divergent evolution of trans-Cinnamaldehyde and Eugenol biosynthesis in Cinnamomum cassia and Cinnamomum verum.

Closely related plant species often exhibit striking diversity in their specialized metabolism, yet the genomic and evolutionary mechanisms driving this divergence are often elusive. The globally important cinnamon species Cinnamomum cassia and C. verum represent a classic example, producing leaf essential oils dominated by trans-cinnamaldehyde (t-CALD) and eugenol, respectively. Here, we combined chromosome-level genome assemblies for both species with comparative transcriptomics and biochemistry to decipher the molecular basis of this metabolic specialization. We show that, while the core phenylpropanoid pathway is largely conserved, the evolutionary innovation lies in the lineage-specific optimization of key terminal enzymes. A combination of enzyme kinetics, structural modelling and molecular dynamics simulations, and site-directed mutagenesis demonstrate that C. cassia possesses a 4-coumarate:CoA ligase (Cc4CL3) with strong substrate preference for trans-cinnamic acid, thus driving efficient t-CALD production. Furthermore, we found that low expression of CCR1 (cinnamoyl-CoA reductase), the key enzyme catalyzing cinnamoyl-CoA to t-CALD, likely limits t-CALD biosynthesis in C. verum. This expression divergence is associated with the presence of short tandem repeats (STRs) in the CcCCR1 promoter, which are absent in CvCCR1. In the eugenol biosynthetic pathway, the coniferyl alcohol acetyltransferase (CFAT) gene family has undergone pronounced expansion in C. verum. Notably, molecular dynamics simulations and site-directed mutagenesis reveal that CvCFAT5 has a deeper substrate-binding pocket than its ortholog in C. cassia, resulting in stable substrate binding and superior catalytic efficiency that drives elevated eugenol synthesis. Our study elucidates how distinct evolutionary trajectories-enzyme optimization and gene family expansion-can direct metabolic flux toward different metabolites in a species-specific manner, providing a genomic resource and a mechanistic framework for understanding metabolic diversification in plants.