Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation  

作　　者：Qing Li Xiaojia Guo Xueying Wang Junting Wang Li Wan Haizhao Xue Zongbao K.Zhao 

机构地区：[1]Laboratory of Biotechnology,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,Liaoning,China [2]University of Chinese Academy of Sciences,Beijing 100049,China [3]State Key Laboratory of Catalysis,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,Liaoning,China [4]Dalian Key Laboratory of Energy Biotechnology,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,Liaoning,China

出　　处：《Journal of Energy Chemistry》2023年第4期31-36,共6页能源化学（英文版）

基　　金：supported by the National Key R&D Program of China(2019YFA0904900);the National Natural Science Foundation of China(21877112,21837002,21721004)。

摘　　要：Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.

关 键 词：Biocatalysis Cytochrome P450 Directed evolution Fatty acid hydroxylation Non-natural cofactor Synthetic biology 

分 类 号：TQ426.97[化学工程]