Engineering of cofactor preference and catalytic activity of methanol dehydrogenase by growth-coupled directed evolution  

作　　者：Jinxing Yang Liwen Fan Guimin Cheng Tao Cai Jibin Sun Ping Zheng Shuang Li Yu Wang 

机构地区：[1]School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,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]College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300222,China

出　　处：《Green Carbon》2024年第2期242-251,共10页绿碳（英文）

基　　金：supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110201);the National Key R&D Program of China(2018YFA0901500);the National Natural Science Foundation of China(32070083 and 32222004);the Innovation Fund of Haihe Laboratory of Synthetic Biology(22HHSWSS00017);the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177);the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008).

摘　　要：Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(MDH)catalyzes the oxidation of methanol to formaldehyde via NADH generation,providing a highly active C1 intermediate and reducing power for subsequent biosynthesis.However,the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization,especially in nicotinamide adenine dinucleotide phosphate(NADP^(+))-dependent biosynthesis.Herein,we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334.NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs,establishing a positive correlation between the cell growth rate and MDH activity.Using this principle,MDH mutants exhibiting a 20-fold improvement in catalytic efficiency(k_(cat)/K_(m))and a 90-fold cofactor specificity switch from NAD^(+)to NADP+without a decrease in specific enzyme activity,were efficiently screened from random and semi-rationally designed libraries.We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)^(+)-dependent enzymes.

关 键 词：Growth-coupled screening Methanol dehydrogenase Cofactor engineering Directed evolution C1 bioconversion 

分 类 号：O64[理学—物理化学]