Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde  

作　　者：Jin Qian Liwen Fan Jinxing Yang Jinhui Feng Ning Gao Guimin Cheng Wei Pu Wenjuan Zhou Tao Cai Shuang Li Ping Zheng Jibin Sun Depei Wang Yu Wang 

机构地区：[1]College of Biotechnology,Tianjin University of Science and Technology,Tianjin,300222,China [2]Haihe Laboratory of Synthetic Biology,Tianjin,300308,China [3]Key Laboratory of Engineering Biology for Low-carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin,300308,China [4]National Technology Innovation Center of Synthetic Biology,Tianjin,300308,China [5]University of Chinese Academy of Sciences,Beijing,100049,China [6]School of Biology and Biological Engineering,South China University of Technology,Guangzhou,510006,China

出　　处：《Synthetic and Systems Biotechnology》2023年第3期386-395,共10页合成和系统生物技术（英文）

基　　金：the National Key Research and Development Program of China(2018YFA0901500);the National Natural Science Foundation of China(32070083 and 32222004);the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177);the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008);the Innovation Fund of Haihe Laboratory of Synthetic Biology.

摘　　要：Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to formaldehyde.Herein,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic activity.The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants.Mdh_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation libraries.The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity.The beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-helices.Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.

关 键 词：Methanol dehydrogenase Formaldehyde biosensor Directed evolution C1 bioconversion Methanol oxidation METHYLOTROPHY 

分 类 号：O64[理学—物理化学]