Functional Expression of Cannabis sativa Polyketide Enzymes in Chlamydomonas reinhardtii Reveals Metabolic Constraints to Olivetolic Acid Accumulation

Plant specialized metabolites are valuable sources of pharmaceuticals, nutraceuticals, and industrial compounds. Transferring their biosynthetic pathways into microbial hosts remains a major challenge, particularly in photosynthetic microalgae. Here, we investigated the ability of the green microalga Chlamydomonas reinhardtii to functionally express Cannabis sativa polyketide biosynthetic enzymes involved in olivetolic acid synthesis, a key intermediate in cannabinoid biosynthesis. Nuclear transformants carrying the C. sativa tetraketide synthase (CsTKS) and olivetolic acid cyclase (CsOAC) genes were generated. Functional enzyme activity was confirmed by in vitro enzymatic assays using total protein extracts. In vitro olivetolic acid production was detected, verified, and quantified by high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry using authentic standards. However, in vivo olivetolic acid and downstream metabolites were not detected in algal biomass. This indicated that functional enzyme expression did not result in productive intracellular pathway flux. Untargeted metabolomic profiling revealed extensive metabolic rewiring and strain-specific metabolic perturbations following pathway introduction. These findings suggest that heterologous pathway expression triggered broad physiological responses. This study demonstrates that plant polyketide enzymes remain catalytically active in C. reinhardtii; however, intrinsic metabolic and cellular constraints limit in vivo metabolite accumulation. These findings provide important insights into the biological barriers that limit the functional implementation of plant polyketide pathways in photosynthetic microalgae C. reinhardtii and identify key targets for future metabolic and cellular engineering.