Lee, Hoe-Suk Park, Jisu Kim, Taejung Min, Huitae Na, Seongsu Park, Soon Young Park, Young Tae Yeon,Young Joo Ham, Jungyeob 2025-04-11T02:00:09Z 2025-04-11T02:00:09Z 2025-04-10 2025-04 https://pubs.kist.re.kr/handle/201004/152258 Introduction: Cannabinoids possess significant therapeutic potential, but their natural chemical diversity derived from plant biosynthesis is limited. Efficient biotransformation processes are required to expand the range of accessible cannabinoids. This study aimed to enhance the selective biosynthesis of cannabigerolic acid (CBGA) and its derivatives with varying aliphatic chain lengths, which serve as key precursors to various cannabinoids. Methods: We employed computational modeling and structure-guided mutagenesis to engineer the aromatic prenyltransferase NphB. Mutants were designed via in silico docking analyses to optimize substrate orientation and catalytic distance. The variants were expressed in E. coli, and their catalytic efficiencies were evaluated through in vivo whole-cell and in vitro enzymatic assays. Products were identified and quantified by UHPLC-MS. Results: Engineered NphB variants exhibited significant 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 also enhanced the synthesis of 3-geranyl orsellinic acid by 1.3-fold. Notably, novel enzymatic activity was identified that enabled the biosynthesis of 3-geranyl-2,4-dihydroxybenzoic acid. Structural analyses revealed that the mutations improved the spatial positioning of aromatic substrates relative to the co-substrate geranyl pyrophosphate. Discussion: This study demonstrates the feasibility of enzyme design to tailor prenyltransferase specificity for the production of diverse CBGA derivatives. These findings lay the groundwork for the microbial production of novel cannabinoids and offer promising potential for the development of scalable biocatalytic systems for therapeutic and industrial applications. English Frontiers Research Foundation Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase Article 10.3389/fbioe.2025.1563708 1 Frontiers in Bioengineering and Biotechnology, v.13 Frontiers in Bioengineering and Biotechnology 13 Y scie scopus 001473142800001 Biotechnology & Applied Microbiology Engineering, Biomedical Biotechnology & Applied Microbiology Engineering Article PRENYLATION SYNTHASE ENZYME CANNABIS-SATIVA aromatic prenyltransferase NphB computational enzyme design engineered E. coli whole cell biocatalytic system cannabinoid cannabigerolic acid derivative