Biosynthesis of Novel Cannabigerolic Acid Derivatives by Engineering the Substrate Specificity of Aromatic Prenyltransferase

Hoe-Suk Lee ¹ Jisu Park ^1,2 Taejung Kim ^2,3 Huitae Min ² Seongsu Na ^1,2 Soon Young Park ⁴ Young-Tae Park ^2,3* Young Joo Yeon ^5* Jungyeob Ham ^2,3,4*

• ¹ Department of Biochemical Engineering, Gangneung–Wonju National University, Gangneung, Republic of Korea
• ² Natural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of Korea
• ³ Division of Natural Product Applied Science, University of Science and Technology (UST), Daejeon, Republic of Korea
• ⁴ NeoCannBio Co., Ltd., Seoul, Republic of Korea
• ⁵ Gangneung–Wonju National University, Gangneung, Republic of Korea

Cannabinoids represent a diverse class of bioactive compounds with significant therapeutic potential for alleviating pain, reducing inflammation, and managing neuropathies. However, the chemical diversity of cannabinoids directly obtainable from the cannabis plant is inherently limited, restricting their broader application. Addressing this limitation requires efficient biotransformation processes to expand the range of cannabinoids available for therapeutic and industrial use. In this study, we aimed to enhance the selective production of cannabigerolic acid (CBGA) and its derivatives, which are key precursors to various cannabinoids, through the computational design and engineering of the aromatic prenyltransferase NphB. Engineered mutants demonstrated remarkable improvements, with triple mutants achieving a 7-fold increase in CBGA production and a 4-fold increase in cannabigerovarinic acid production. Additionally, a single mutant exhibited a modest 1.3-fold improvement in the synthesis of 3-geranyl orsellinic acid. Notably, we identified novel enzymatic activity for the biosynthesis of 3-geranyl 2,4-dihydroxybenzoic acid. Structural analysis revealed that these mutations optimized the spatial positioning of aromatic substrates relative to the co-substrate geranyl pyrophosphate, facilitating the efficient biosynthesis of diverse CBGA derivatives. This study underscores the potential of enzyme engineering to expand the cannabinoid repertoire, providing a foundation for novel therapeutic and industrial applications.