Abstract Description

Cannabidiolic acid synthase (CBDAS) catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into cannabidiolic acid (CBDA), a key reaction in the cannabinoid biosynthetic pathway. Although cannabinoids are of considerable therapeutic and commercial interest, the structural and biochemical properties of CBDAS enzymes from distinct Cannabis sativa landraces remain underexplored. In this study, CBDAS from the African Swazi Gold landrace, lacking the native signal peptide, was cloned into the pPICZαA vector and expressed in Komagataella phaffii under methanol induction (0.5 % v/v) at 16 °C in Buffered Minimal Methanol-complex Medium (BMMY). The recombinant enzyme, secreted into the culture medium, was purified by immobilized metal affinity chromatography (IMAC) and subjected to structural and functional analyses. Far-UV circular dichroism indicated a secondary structure enriched in α-helices, consistent with a flavoprotein oxidase architecture. Intrinsic tryptophan fluorescence revealed a blue shift upon CBGA binding, suggestive of ligand-induced conformational rearrangements in the active site. Substrate conversion assays demonstrated efficient catalytic conversion of CBGA to CBDA, confirming enzyme functionality. These findings provide the first structural and biochemical characterization of a CBDAS enzyme from any African landrace and establish a framework for comparative analyses of landrace-derived enzymes and for future efforts in cannabinoid pathway engineering in microbial hosts.