Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase
IntroductionCannabinoids 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 biosynthes...
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Frontiers Media S.A.
2025-04-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1563708/full |
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| author | Hoe-Suk Lee Jisu Park Jisu Park Taejung Kim Taejung Kim Huitae Min Seongsu Na Seongsu Na Soon Young Park Young-Tae Park Young-Tae Park Young Joo Yeon Jungyeob Ham Jungyeob Ham Jungyeob Ham |
| author_facet | Hoe-Suk Lee Jisu Park Jisu Park Taejung Kim Taejung Kim Huitae Min Seongsu Na Seongsu Na Soon Young Park Young-Tae Park Young-Tae Park Young Joo Yeon Jungyeob Ham Jungyeob Ham Jungyeob Ham |
| author_sort | Hoe-Suk Lee |
| collection | DOAJ |
| description | IntroductionCannabinoids 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.MethodsWe 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.ResultsEngineered 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.DiscussionThis 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. |
| format | Article |
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| institution | OA Journals |
| issn | 2296-4185 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj-art-e4805264ae544000a8b2bf0a290d40862025-08-20T02:16:50ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852025-04-011310.3389/fbioe.2025.15637081563708Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferaseHoe-Suk Lee0Jisu Park1Jisu Park2Taejung Kim3Taejung Kim4Huitae Min5Seongsu Na6Seongsu Na7Soon Young Park8Young-Tae Park9Young-Tae Park10Young Joo Yeon11Jungyeob Ham12Jungyeob Ham13Jungyeob Ham14Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of KoreaDepartment of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of KoreaNatural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of KoreaNatural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of KoreaDivision of Natural Product Applied Science, University of Science and Technology (UST), Daejeon, Republic of KoreaNatural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of KoreaDepartment of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of KoreaNatural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of KoreaNeoCannBio Co., Ltd., Seoul, Republic of KoreaNatural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of KoreaDivision of Natural Product Applied Science, University of Science and Technology (UST), Daejeon, Republic of KoreaDepartment of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of KoreaNatural Product Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Republic of KoreaDivision of Natural Product Applied Science, University of Science and Technology (UST), Daejeon, Republic of KoreaNeoCannBio Co., Ltd., Seoul, Republic of KoreaIntroductionCannabinoids 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.MethodsWe 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.ResultsEngineered 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.DiscussionThis 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.https://www.frontiersin.org/articles/10.3389/fbioe.2025.1563708/fullcannabinoidcannabigerolic acid derivativearomatic prenyltransferase NphBcomputational enzyme designengineered E. coli whole cellbiocatalytic system |
| spellingShingle | Hoe-Suk Lee Jisu Park Jisu Park Taejung Kim Taejung Kim Huitae Min Seongsu Na Seongsu Na Soon Young Park Young-Tae Park Young-Tae Park Young Joo Yeon Jungyeob Ham Jungyeob Ham Jungyeob Ham Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase Frontiers in Bioengineering and Biotechnology cannabinoid cannabigerolic acid derivative aromatic prenyltransferase NphB computational enzyme design engineered E. coli whole cell biocatalytic system |
| title | Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase |
| title_full | Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase |
| title_fullStr | Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase |
| title_full_unstemmed | Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase |
| title_short | Biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase |
| title_sort | biosynthesis of novel cannabigerolic acid derivatives by engineering the substrate specificity of aromatic prenyltransferase |
| topic | cannabinoid cannabigerolic acid derivative aromatic prenyltransferase NphB computational enzyme design engineered E. coli whole cell biocatalytic system |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2025.1563708/full |
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