Targeted phytocannabinomics provides new insights into the biosynthetic origin of cis-Δ⁹-THCA in Cannabis sativa L

Recent studies have reported the occurrence of cis-Δ⁹-tetrahydrocannabinol (cis-Δ⁹-THC) and its carboxylated precursor, cis-Δ⁹-tetrahydrocannabinolic acid (cis-Δ⁹-THCA), minor isomers of the well-known trans counterparts. However, their origin remains unclear and several hypotheses have been proposed. In this work, cis-Δ⁹-THCA and the major phytocannabinoids, including cannabidiolic acid (CBDA), trans-Δ⁹-tetrahydrocannabinolic acid (trans-Δ⁹-THCA), cannabichromenic acid (CBCA), and their biosynthetic precursor cannabigerolic acid (CBGA), were quantified in a large and diverse set of C. sativa accessions using a targeted metabolomics approach based on liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (HPLC-HRMS). Our findings indicate that cis-Δ⁹-THCA does not appear to be chemically derived from other cannabinoids, prompting new considerations regarding its biosynthetic origin. Notably, growth-stage analyses revealed a parallel accumulation pattern between cis-Δ⁹-THCA and CBCA, suggesting the potential involvement of CBCA synthase in its formation. Overall, this study provides new evidence on the distribution, variability, and possible biosynthetic pathways of cis-Δ⁹-THCA, enriching current understanding of cannabinoid diversity in C. sativa.