| Abstract | The psychoactive and analgesic cannabinoids (e.g. Δ9-tetrahydrocannabinol, THC) in Cannabis sativa are formed from the short-chain fatty acyl-CoA precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g−1 fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes encoding putative AAEs, including CsAAE1 which shows high-transcript abundance in glandular trichomes. In vitro assays show that recombinant CsAAE1 activates hexanoate and other short- and medium-chain fatty acids. This activity and the trichome-specific expression of CsAAE1 suggests that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis shows that CsAAE1 groups with peroxisomal AAEs, it lacks a peroxisome targeting sequence 1 (PTS1) and localizes to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome EST dataset for enzymes of fatty acid metabolism. The high abundance of transcripts encoding desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway involving the oxygenation and breakdown of unsaturated fatty acids. |
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