Rational Design and Modification of NphB for Cannabinoids Biosynthesis
Wenhao Xia,
Shimeng Liu,
Huanyu Chu,
Xianqing Chen,
Lihui Huang,
Tao Bai,
Xi Jiao,
Wen Wang,
Huifeng Jiang,
Xiao Wang
Affiliations
Wenhao Xia
New Cornerstone Science Laboratory, Shaanxi Key Laboratory of Qinling Ecological Intelligent Monitoring and Protection, School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710072, China
Shimeng Liu
Jiaxing Synbiolab Technology Co., Ltd., Jiaxing 314000, China
Huanyu Chu
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Xianqing Chen
New Cornerstone Science Laboratory, Shaanxi Key Laboratory of Qinling Ecological Intelligent Monitoring and Protection, School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710072, China
Lihui Huang
Jiaxing Synbiolab Technology Co., Ltd., Jiaxing 314000, China
Tao Bai
Jiaxing Synbiolab Technology Co., Ltd., Jiaxing 314000, China
Xi Jiao
Jiaxing Synbiolab Technology Co., Ltd., Jiaxing 314000, China
Wen Wang
New Cornerstone Science Laboratory, Shaanxi Key Laboratory of Qinling Ecological Intelligent Monitoring and Protection, School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710072, China
Huifeng Jiang
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Xiao Wang
Jiaxing Synbiolab Technology Co., Ltd., Jiaxing 314000, China
The rapidly growing field of cannabinoid research is gaining recognition for its impact in neuropsychopharmacology and mood regulation. However, prenyltransferase (NphB) (a key enzyme in cannabinoid precursor synthesis) still needs significant improvement in order to be usable in large-scale industrial applications due to low activity and limited product range. By rational design and high-throughput screening, NphB’s catalytic efficiency and product diversity have been markedly enhanced, enabling direct production of a range of cannabinoids, without the need for traditional enzymatic conversions, thus broadening the production scope of cannabinoids, including cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerovarin (CBGV), and cannabigerovarinic acid (CBGVA). Notably, the W3 mutant achieved a 10.6-fold increase in CBG yield and exhibited a 10.3- and 20.8-fold enhancement in catalytic efficiency for CBGA and CBGV production, respectively. The W4 mutant also displayed an 9.3-fold increase in CBGVA activity. Molecular dynamics simulations revealed that strategic reconfiguration of the active site’s hydrogen bonding network, disulfide bond formation, and enhanced hydrophobic interactions are pivotal for the improved synthetic efficiency of these NphB mutants. Our findings advance the understanding of enzyme optimization for cannabinoid synthesis and lay a foundation for the industrial-scale production of these valuable compounds.