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 s...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.展开更多
Carbon dioxide fixation presents a potential solution for mitigating the greenhouse gas issue.During carbon dioxide fixation,C1 compound reduction requires a high energy supply.Thermodynamic calculations suggest that ...Carbon dioxide fixation presents a potential solution for mitigating the greenhouse gas issue.During carbon dioxide fixation,C1 compound reduction requires a high energy supply.Thermodynamic calculations suggest that the energy source for cofactor regeneration plays a vital role in the effective enzymatic C1 reduction.Hydrogenase utilizes renewable hydrogen to achieve the regeneration and supply cofactor nicotinamide adenine dinucleotide(NADH),providing a driving force for the reduction reaction to reduce the thermodynamic barrier of the reaction cascade,and making the forward reduction pathway thermodynamically feasible.Based on the regeneration of cofactor NADH by hydrogenase,and coupled with formaldehyde dehydrogenase and formolase,a favorable thermodynamic mode of the C1 reduction pathway for reducing formate to dihydroxyacetone(DHA)was designed and constructed.This resulted in accumulation of 373.19μmol·L^(-1) DHA after 2 h,and conversion reaching 7.47%.These results indicate that enzymatic utilization of hydrogen as the electron donor to regenerate NADH is of great significance to the sustainable and green development of bio-manufacturing because of its high economic efficiency,no by-products,and environment-friendly operation.Moreover,formolase efficiently and selectively fixed the intermediate formaldehyde(FALD)to DHA,thermodynamically pulled formate to efficiently reduce to DHA,and finally stored the low-grade renewable energy into chemical energy with high energy density.展开更多
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)+3 种基金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).
文摘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.
基金funded by the National Key Research and Development Program of China(Grant No.2022YFC2105900)the National Natural Science Foundation of China(Grant Nos.22378015 and 52073022).
文摘Carbon dioxide fixation presents a potential solution for mitigating the greenhouse gas issue.During carbon dioxide fixation,C1 compound reduction requires a high energy supply.Thermodynamic calculations suggest that the energy source for cofactor regeneration plays a vital role in the effective enzymatic C1 reduction.Hydrogenase utilizes renewable hydrogen to achieve the regeneration and supply cofactor nicotinamide adenine dinucleotide(NADH),providing a driving force for the reduction reaction to reduce the thermodynamic barrier of the reaction cascade,and making the forward reduction pathway thermodynamically feasible.Based on the regeneration of cofactor NADH by hydrogenase,and coupled with formaldehyde dehydrogenase and formolase,a favorable thermodynamic mode of the C1 reduction pathway for reducing formate to dihydroxyacetone(DHA)was designed and constructed.This resulted in accumulation of 373.19μmol·L^(-1) DHA after 2 h,and conversion reaching 7.47%.These results indicate that enzymatic utilization of hydrogen as the electron donor to regenerate NADH is of great significance to the sustainable and green development of bio-manufacturing because of its high economic efficiency,no by-products,and environment-friendly operation.Moreover,formolase efficiently and selectively fixed the intermediate formaldehyde(FALD)to DHA,thermodynamically pulled formate to efficiently reduce to DHA,and finally stored the low-grade renewable energy into chemical energy with high energy density.
