Enhanced chemoselectivity of a plant cytochrome P450 through protein engineering of surface and catalytic residues  

作　　者：Xiaopeng Zhang Wei Luo Yinying Yao Xuming Luo Chao Han Yang Zhong Bo Zhang Dawei Li Lida Han Sanwen Huang Per Greisen Yi Shang 

机构地区：[1]Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture,Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518116,China [2]Yunnan Key Laboratory of Potato Biology,The CAAS-YNNU-YINMORE Joint Academy of Potato Sciences,Yunnan Normal University,Kunming 650500,China [3]National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research,Huazhong Agricultural University,Wuhan 430070,China [4]Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China [5]Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of Ministry of Agriculture,Sino-Dutch Joint Lab of Horticultural Genomics,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China [6]Novo Nordisk Research Center Seattle Inc,Seattle,WA 98109,USA

出　　处：《aBIOTECH》2021年第3期215-225,共11页生物技术通报（英文版）

基　　金：the National Key Research and Development Program of China(2018YFA0901800);Yunnan Science Fund(202005AE160015 and 2019FJ004);This work was also supported from Shenzhen Municipal Governments.

摘　　要：Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their functions.We previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these enzymes.With this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active site.Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells,expressing them in heterologous hosts usually results in problems of expression or activity.Here,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active site.After screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of CYP87D20.In addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis.This study shows the importance of considering the interplay between surface and active site residues for P450 engineering.Our integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.

关 键 词：Plant cytochrome P450 Protein engineering ROSETTA Amino acid co-evolution Surface residue 

分 类 号：Q94[生物学—植物学]