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Enhanced chemoselectivity of a plant cytochrome p450 through protein engineering of surface and catalytic residues

aBIOTECH 2021年第3期2卷 215-225页

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 Shenzhen Branch Guangdong Laboratory of Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518116China Yunnan Key Laboratory of potato Biology The CAAS-YNNU-YINMORE Joint Academy of Potato SciencesYunnan Normal UniversityKunming 650500China National Key Laboratory of Crop Genetic Improvement and National Centre of plant Gene Research Huazhong Agricultural UniversityWuhan 430070China Biotechnology Research Institute Chinese Academy of Agricultural SciencesBeijing 100081China Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of Ministry of Agriculture Sino-Dutch Joint Lab of Horticultural GenomicsInstitute of Vegetables and FlowersChinese Academy of Agricultural SciencesBeijing 100081China Novo Nordisk Research Center Seattle Inc SeattleWA 98109USA

cytochrome p450s(p450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse *** 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 *** 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 *** this strategy,we repurposed a multi-functional p450(CYp87D20)into a monooxygenase after red esigning its active *** 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 ***,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active *** screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of *** 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 *** study shows the importance of considering the interplay between surface and active site residues for p450 *** 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

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Engineering plant cytochrome p450s for Enhanced Synthesis of Natural products: past Achievements and Future perspectives

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plant Communications 2020年第1期1卷 57-63页

作者： Yi Shang Sanwen Huang Key Laboratory for potato Biology of Yunnan province The CAAS-YNNU-YINMORE Joint Academy of Potato ScienceYunnan Normal UniversityKunmingChina Lingnan Guangdong Laboratory of Modern Agriculture Genome Analysis Laboratory of the Ministry of AgricultureAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhenChina

cytochrome p450s(p450s)are the most versatile catalysts and are widely used by plants to synthesize a vast array of structurally diverse specialized metabolites that not only play essential ecological roles but also constitute a valuable resource for the development of new *** accelerate the metabolic engineering of these high-value metabolites,it is imperative to identify and characterize pathway p450s,and to further improve their activities through protein *** this review,we focus on p450 engineering and summarize the major strategies for enhancing the stability and activity of p450s and successful cases of p450 *** in which the functions of p450s were altered to create de novo metabolic pathways or novel compounds are discussed as *** also overview emerging tools,specifically DNA synthesis,machine learning,and de novo protein design,as well as the evolutionary patterns of p450s unveiled from a massive number of DNA sequences that could be integrated to accelerate the engineering of these *** approaches would greatly aid in the exploitation of plant-specialized metabolites or derivatives for various uses including medical applications.

关键词： plant cytochrome p450 plant natural product protein engineering amino acid co-evolution protein design

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