Insights into cannabinoid biosynthesis in Chlamydomonas reinhardtii : successes with NphB and limitations of CBDAS expression

The growing legalization of Cannabis has increased demand for cannabinoids (CBs). Currently, pharmaceutically relevant CBs are primarily extracted from Cannabis, a process that presents challenges related to yield variability and purity. To overcome these limitations, microbial platforms are being explored for the sustainable production of specific CBs. Compared to conventional microbial hosts, the photosynthetic microalga Chlamydomonas reinhardtii offers plant-like post-translational modifications, low-cost cultivation, CO2 fixation, and low endotoxin contamination, making it a potential chassis for CB biosynthesis. Here, we expressed a codon-optimized, soluble aromatic prenyltransferase (NphBG286S/Y288A) from Streptomyces and Cannabis sativa cannabidiolic acid synthase (CBDAS) in the nuclear genome of C. reinhardtii; transformants were screened for integration, gene expression, protein accumulation, enzymatic activity, and CB production. Our results provide evidence of functional NphB expression in C. reinhardtii, with in vitro CBGA production reaching up to 633 {+/-} 58 {micro}g/L. Although CBDAS transcripts were detected under multiple construct designs, neither protein nor CBDA was accumulated, suggesting limitations in expression, localization, or post-translational processing in C. reinhardtii. Our study provides the first demonstration of in vitro CBGA biosynthesis in the photosynthetic model microalga C. reinhardtii, highlighting its potential as a future platform for CB production. It also highlights key challenges in nuclear expression of plant-derived enzymes, such as CBDAS, emphasizing that improved regulatory control, subcellular targeting, and mRNA processing will be required to achieve full pathway reconstruction in C. reinhardtii. HighlightsO_LIFirst report of active NphB expression in Chlamydomonas reinhardtii C_LIO_LINphB exhibits measurable in vitro activity, producing cannabigerolic acid in algal extracts C_LIO_LIEnzyme localization, construct design, and substrate availability critically influence functionality. C_LI