Evaluation of the Cannabidiolic Acid Synthase (CBDAS) variant’s activity from hemp in transgenic Nicotiana benthamiana plants.
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Date
2025
Authors
Shujat, Salma
University of Lethbridge. Faculty of Arts and Science
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Lethbridge, Alta. : University of Lethbridge, Dept. of Biological Sciences
Abstract
Cannabis sativa L., historically controversial, has gained economic and scientific significance in Canada following legalization, primarily due to its primary cannabinoids: cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC). High-THC cannabis serves recreational and medicinal purposes, while high-CBD cultivars are increasingly valued in medical and cosmetic industries. Hemp, legally defined as cannabis containing less than 0.3% THC, is widely used in food, textiles, and biodegradable materials. Both cannabidiol and delta-9-tetrahydrocannabinol are synthesized through decarboxylation of their acidic precursors, cannabidiolic acid (CBDA) and delta-9-tetrahydrocannabinolic acid (THCA), respectively, which are derived from a common precursor, cannabigerolic acid (CBGA), through the enzymatic action of cannabidiolic acid synthase (CBDAS) and delta-9-tetrahydrocannabinolic acid synthase (THCAS). These enzymes exhibit partial promiscuity, meaning they can convert CBGA into multiple cannabinoids, including CBDA, THCA, and cannabichromenic acid (CBCA), typically in ratios ranging from 10:1:1 to 20:1:1. Thus, even in plants lacking the THCAS gene, trace amounts of THCA can be produced, potentially complicating regulatory classification under Health Canada’s guidelines. Despite their structural similarities, bioinformatic analyses have identified unique functional variants of the CBDAS enzyme with differing specificity and activity.
This study evaluated four CBDAS variants, Del_1_108, X59_1_117, Joe_1_129, and CRS1_105, by expressing them in Nicotiana benthamiana via stable genetic transformation. After optimizing assay conditions (including incubation time, temperature, and buffer composition), only Joe_1_129 and X59_1_117 showed enzymatic activity. Both variants catalyzed the exclusive conversion of CBGA to CBDA, with no production of THCA or CBCA, indicating enhanced specificity. Extended incubation (12–16 hours) further improved enzyme efficiency. While Joe_1_129 demonstrated higher conversion efficiency (2.46%), X59_1_117 exhibited better catalytic performance (1.14 vs. 0.89), suggesting functional specialization.
These results provide valuable insights into the evolution and function of CBDAS enzymes and support metabolic engineering strategies aimed at producing hemp cultivars with high CBDA and negligible THC content. Such advances have practical implications for pharmaceutical, cosmetic, and industrial applications, and future research integrating genomic, transcriptomic, and metabolomic approaches could further refine cannabinoid biosynthesis pathways.