Extrachromosomal expression of functional Cannabis sativa cannabidiolic acid synthase in Phaedodactylum tricornutum

Cannabis sativa 's cannabidiolic acid (CBDA) offers significant therapeutic potential without inducing psychotropic effects but is typically found as part of a complex mixture of metabolites in plant extracts. Using a heterologous expression platform could allow the production of pure CBDA. Here, we propose to express CBDA synthase (CBDAS) in Phaeodactylum tricornutum . Episomes carrying CBDAS variants, incorporating the native signal peptide (CBDAS) or the highly abundant secreted protein 1 secretory signal peptide (SP:CBDAS) were constructed. CBDAS variants were tagged with the yellow fluorescent protein (YFP), introduced into the marine diatom, and screened by fluorescence. Confocal microscopy revealed that CBDAS and SP:CBDAS arranged in aggregated structures indicative of secretory pathway involvement. Western blot assays confirmed whole construct accumulation intracellularly, while soluble YFP was detected extracellularly. Finally, enzymatic assays showed CBDA production by both CBDAS and SP:CBDAS strains, confirming the potential of P. tricornutum as a platform for cannabinoid biosynthesis. • Microalgae production of cannabidiolic acid (CBDA), a bioactive cannabinoid (CB) • Promising photosynthetic platform for sustainable biomanufacturing pharmaceuticals • Cannabis CBDA synthase was successfully heterologously expressed in Phaeodactylum tricornutum . • Fluorescent tagging and microscopy used to track CBDA synthase localization. • Secretory signal peptides enhance CBDA synthase targeting and secretion pathways.