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Directed Evolution of P450 Fatty Acid Decarboxylases via High‐Throughput Screening towards Improved Catalytic Activity
Author(s) -
Xu Huifang,
Liang Weinan,
Ning Linlin,
Jiang Yuanyuan,
Yang Wenxia,
Wang Cong,
Qi Feifei,
Ma Li,
Du Lei,
Fourage Laurent,
Zhou Yongjin J.,
Li Shengying
Publication year - 2020
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201901347
Subject(s) - directed evolution , dna shuffling , high throughput screening , mutagenesis , chemistry , escherichia coli , metabolic engineering , substrate (aquarium) , biochemistry , combinatorial chemistry , catalysis , fatty acid , mutant , enzyme , biology , gene , ecology
Abstract P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one‐step direct production of industrially important 1‐alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high‐throughput screening (HTS) method. Here, we devise a catalase‐deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H 2 O 2 ‐consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium‐chain 1‐alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H 2 O 2 as co‐substrate.