Biochemical Investigations of Bibenzyl Cannabinoids from the Liverwort Radula

Abstract

Phytocannabinoids, plant-derived molecules characterized by a resorcinyl core structure and its bioactivity for binding and activating endocannabinoid receptors, were believed to be exclusive to Cannabis sativa until the discovery of perrottetinene in the genus Radula. This bibenzyl phytocannabinoid exhibits unique pharmacological effects in mammalian models, highlighting its potential value. However, its biosynthetic pathway remains elusive to date. Using the well-studied phytocannabinoid biosynthetic pathway of C. sativa as a reference, key candidate enzymes involved in perrottetinene biosynthesis were identified. Of primary interest in this study is a putative type III polyketide synthase (PKS) responsible for assembling the core phytocannabinoid structure. Type III PKSs catalyze the formation of many valuable plant metabolites, including flavonoids, curcumin, and cannabidiol. Seven candidate Radula PKS genes were identified through homology and transcriptomic analysis and functionally characterized in in vivo conditions by heterologous expression in Saccharomyces cerevisiae strains engineered to produce various acyl-CoA precursors. Enzymatic activity with both native and non-native substrates was assessed using liquid chromatography mass-spectrometry (LC-MS). The characterization of these novel PKSs provides a foundation for the bioengineering of perrottetinene and synthetic cannabinoids with potential pharmaceutical and industrial applications. To further facilitate gene characterization and metabolic engineering, a CRISPR-Cas9 multiplex genome editing platform was developed in S. cerevisiae. This system, termed 4×4, enables the sequential integration of up to 16 unique DNA fragments by four sequential integrations of a set of four genes. The platform allows for the rapid construction of complex biosynthetic pathways, making it a powerful tool for bioengineering the perrottetinene biosynthetic pathway and for leveraging yeast as a cell factory for the production of high-value metabolites.