Cannabinoids are high-value bioactive compounds whose sustainable production remains challenging, prompting interest in biocatalytic and microbial platforms as alternatives to plant extraction. In this study, we investigated the heterologous expression and functionality of two key cannabinoid-related enzymes in the photosynthetic microalga
Chlamydomonas reinhardtii: the aromatic prenyltransferase, NphB
G286S/Y288A from
Streptomyces sp., and the plant-derived cannabidiolic acid synthase (CBDAS) from
Cannabis sativa. Codon-optimized genes were introduced into the nuclear genome of
C. reinhardtii using several construct configurations and promoters, and stable transformants were generated and characterized for genomic integration, transcript accumulation, protein production, enzymatic activity, and cannabinoid-related metabolite formation. While NphB protein accumulation was achieved under the
PSAD promoter control, CBDAS was not detected at the protein level under any condition tested. In vitro enzymatic assays using soluble algal protein extracts from
NphB-expressing lines confirmed catalytic activity, yielding cannabigerolic acid (CBGA), reaching up to 633 ± 58 µg L
−1. However, no CBGA production was detected in vivo, despite substrate supplementation. These results indicate that, although bacterial prenyltransferase can be functionally expressed in
C. reinhardtii, efficient metabolic conversion in vivo is limited by cellular and biochemical constraints, including substrate availability, intracellular compartmentalization, and potential competition with endogenous pathways. In contrast, the absence of detectable CBDAS highlights the challenges associated with expressing complex plant oxidocyclases in this photosynthetic host. Overall, this work provides mechanistic insights into enzyme compatibility and metabolic bottlenecks in microalgal systems and outlines key considerations for the future development of photosynthetic platforms for cannabinoid biocatalysis.
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