A structural and data-driven approach to engineering a plant cytochrome P450 enzyme  

作　　者：Dawei Li Yongshuo Ma Yuan Zhou Junbo Gou Yang Zhong Lingling Zhao Lida Han Sergey Ovchinnikov Ling Ma Sanwen Huang Per Greisen Yi Shang 

机构地区：[1]Agricultural Genome Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518124,China [2]School of Pharmacy,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China [3]Institute of Apicultural Research,Chinese Academy of Agricultural Sciences,Beijing 100093,China [4]Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China [5]FAS Center for Systems Biology,Harvard University,Cambridge MA 02138,USA [6]The CAAS-YNNU-YIN MO RE Joint Academy of Potato Science,Yunnan Normal University,Yunnan 650500,China [7]Global Research,Novo Nordisk A/S,Malov DK-2760,Denmark

出　　处：《Science China(Life Sciences)》2019年第7期873-882,共10页中国科学（生命科学英文版）

基　　金：supported by the National Natural Science Foundation of China(31672171,81773597);Shenzhen municipal(JCYJ20160530191729620 to Y.S.)Dapeng district governments

摘　　要：Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products.Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities.This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s.However,the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging,often because of a lack of high-throughput screening methods and crystal structures of P450 s.In this study,we used an integrated approach combining computational protein design,evolutionary information,and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber.After three rounds of iterative design and evaluation of 96 protein variants,CYP87 D20,which is involved in the cucurbitacin C biosynthetic pathway,was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol.This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol,the precursor of the natural sweetener mogroside,or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s. However, the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450 s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87 D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.

关 键 词：plant P450 ENGINEERING protein design ROSETTA amino acid CO-EVOLUTION CUCURBITACIN MOGROSIDE 

分 类 号：Q[生物学]