Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion  

作　　者：Guimin Cheng Hongbing Sun Qian Wang Jinxing Yang Jing Qiao Cheng Zhong Tao Cai Yu Wang 

机构地区：[1]College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300222,People’s Republic of 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,People’s Republic of China [4]School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China [5]National Center of Technology Innovation for Synthetic Biology,Tianjin 300308,China

出　　处：《Bioresources and Bioprocessing》2024年第1期655-661,共7页生物资源与生物加工(英文)

基　　金：supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110201);the National Natural Science Foundation of China(32222004 and 32070083);the Major Program and Innovation Fund of Haihe Laboratory of Synthetic Biology(22HHSWSS00003 and 22HHSWSS00017);the CAS Project for Young Scientists in Basic Research(YSBR-072);the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177);the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008).

摘　　要：Background Formolase(FLS)is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone(DHA).FLS lays the foundation for several artificial carbon fixation and valorization pathways,such as the artificial starch anabolic pathway.However,the application of FLS is limited by its low catalytic activity and product promiscuity.Findings FLS,designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida,was selected as a candidate for modification.To evaluate its catalytic activity,25 residues located within an 8Ådistance from the active center were screened using single-point saturation mutagenesis.A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants.After screening approximately 5,000 variants(approximately 200 transformants per site),several amino acid sites that were not identified by directed evolution were found to improve DHA formation.The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold.Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site,predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation.Conclusions This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency,which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.

关 键 词：Formolase DIHYDROXYACETONE C1 bioconversion Carbon fixation Synthetic methylotrophy 

分 类 号：R59[医药卫生—内科学]