TPS-g structural divergence drives high-purity linalool accumulation in Cinnamomum camphora var. linaloolifera

Cinnamomum camphora var. linaloolifera is a primary source of natural linalool, an acyclic monoterpene with high industrial value, yet the molecular basis of its high-purity accumulation remains poorly understood. In this study, we characterized 'Nan'an 1', an elite cultivar, identifying it as a unique chemotype with exceptional linalool purity (88.30%) and negligible metabolic byproducts via comparative metabolomics. Genome-wide analysis identified 46 CcTPS genes, revealing a significant expansion of the TPS-b subfamily. Notably, the TPS-g subfamily clustered closely with TPS-b but exhibited a specific loss of the cyclization-associated RRX8W motif. This structural divergence provides a theoretical basis for the functional specialization of TPS-g in acyclic monoterpene biosynthesis. Transcriptomic and qRT-PCR analyses revealed that the high expression of four TPS-g candidates (CcTPS14, CcTPS15, CcTPS16, and CcTPS32) significantly correlates with linalool accumulation. Furthermore, heterologous expression in Escherichia coli and in vitro enzymatic assays conclusively demonstrated that these four recombinant proteins function as highly specific linalool synthases, efficiently converting geranyl diphosphate (GPP) into linalool. These findings suggest that the TPS-g subfamily likely originated from the TPS-b lineage through the specific loss of the RRX8W domain, thereby specializing in linalool synthesis. This study elucidates the genetic and molecular mechanisms of high-purity linalool accumulation, offering precise target genes for metabolic engineering.