标题：Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening towards Improved Catalytic Activity
作者：Xu, Huifang; Liang, Weinan; Ning, Linlin; Jiang, Yuanyuan; Yang, Wenxia; Wang, Cong; Qi, Feifei; Ma, Li; Du, Lei; Fourage, Laurent; 更多 作者机构：[Xu, Huifang; Liang, Weinan; Ning, Linlin; Jiang, Yuanyuan; Yang, Wenxia; Wang, Cong; Qi, Feifei; Ma, Li; Du, Lei; Li, Shengying] Chinese Acad Sci, Qi 更多
通讯作者：Li, SY;Li, SY;Li, SY
通讯作者地址：[Li, SY]Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, CAS Key Lab Biofuels, Shandong Prov Key Lab Synthet Biol, Qingdao 266101, Shandong 更多
关键词：1-alkene; directed evolution; high-throughput screening; H2O2; P450; fatty acid decarboxylases
摘要：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 H2O2-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 H2O2 as co-substrate.