Recycling greenhouse gases from industrial emissions is necessary for a genuine circular carbon economy.One-carbon(C1)compounds like methanol produced from greenhouse gases and its subsequent use as a feedstock hold g...Recycling greenhouse gases from industrial emissions is necessary for a genuine circular carbon economy.One-carbon(C1)compounds like methanol produced from greenhouse gases and its subsequent use as a feedstock hold great promise in driving the next generation of biomanufacturing.This review explores the emerging field of synthetic methylotrophy,which focuses on engineering microbial cell factories to convert methanol into useful bioproducts like industrial chemicals,pharmaceuti-cals,fuels,and food.Native methylotrophs have natural pathways for methanol utilization,but obstacles such as metabolic inefficiency and the availability of genetic modification tools limit their use.In contrast,Synthetic methylotrophy makes use of model organisms such as Escherichia coli and Saccharomyces cerevisiae,which can be genetically altered to enhance the efficiency of bioconversion and methanol utilization.Although formaldehyde detoxification and enzyme optimization have improved recently due to developments in metabolic engineering,there are still many obstacles to overcome,such as limited methanol uptake and toxicity problems.The recent developments in synthetic methylotrophy are highlighted in this review,which also stresses the necessity of integrating advanced synthetic biology techniques and performing further research into metabolic pathways of methanol assimilation.Together with a consideration of the techno-economic aspects affecting the scalability of these novel processes,the potential for C1-based biomanufacturing to support sustainable production methods is emphasized.展开更多
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
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...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.展开更多
One-carbon compounds,such as methanol,are becoming potential alternatives to sugars as feedstocks for the biological production of chemicals,fuels,foods,and pharmaceuticals.Efficient biological production often requir...One-carbon compounds,such as methanol,are becoming potential alternatives to sugars as feedstocks for the biological production of chemicals,fuels,foods,and pharmaceuticals.Efficient biological production often requires extensive genetic manipulation of a microbial host strain,making well-characterised and geneticallytractable model organisms like the yeast Saccharomyces cerevisiae attractive targets for the engineering of methylotrophic metabolism.S.cerevisiae strains S288C and CEN.PK are the two best-characterised and most widely used hosts for yeast synthetic biology and metabolic engineering,yet they have unpredictable metabolic phenotypes related to their many genomic differences.We therefore sought to benchmark these two strains as potential hosts for engineered methylotrophic metabolism by comparing their growth and transcriptomic responses to methanol.CEN.PK had improved growth in the presence of methanol relative to the S288C derivative BY4741.The CEN.PK transcriptome also had a specific and relevant response to methanol that was either absent or less pronounced in the BY4741 strain.This response included up-regulation of genes associated with mitochondrial and peroxisomal metabolism,alcohol and formate dehydrogenation,glutathione metabolism,and the global transcriptional regulator of metabolism MIG3.Over-expression of MIG3 enabled improved growth in the presence of methanol,suggesting that MIG3 is a mediator of the superior CEN.PK strain growth.CEN.PK was therefore identified as a superior strain for the future development of synthetic methylotrophy in S.cerevisiae.展开更多
文摘Recycling greenhouse gases from industrial emissions is necessary for a genuine circular carbon economy.One-carbon(C1)compounds like methanol produced from greenhouse gases and its subsequent use as a feedstock hold great promise in driving the next generation of biomanufacturing.This review explores the emerging field of synthetic methylotrophy,which focuses on engineering microbial cell factories to convert methanol into useful bioproducts like industrial chemicals,pharmaceuti-cals,fuels,and food.Native methylotrophs have natural pathways for methanol utilization,but obstacles such as metabolic inefficiency and the availability of genetic modification tools limit their use.In contrast,Synthetic methylotrophy makes use of model organisms such as Escherichia coli and Saccharomyces cerevisiae,which can be genetically altered to enhance the efficiency of bioconversion and methanol utilization.Although formaldehyde detoxification and enzyme optimization have improved recently due to developments in metabolic engineering,there are still many obstacles to overcome,such as limited methanol uptake and toxicity problems.The recent developments in synthetic methylotrophy are highlighted in this review,which also stresses the necessity of integrating advanced synthetic biology techniques and performing further research into metabolic pathways of methanol assimilation.Together with a consideration of the techno-economic aspects affecting the scalability of these novel processes,the potential for C1-based biomanufacturing to support sustainable production methods is emphasized.
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the National Key Research and Development Program of China(2018YFA0901500)the National Natural Science Foundation of China(32070083 and 32222004)+2 种基金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.
文摘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.
基金The Synthetic Biology initiative at Macquarie University is financially supported by an internal grant from the University,and external grants from Bioplatforms Australia,the New South Wales(NSW)Chief Scientist and Engineer,and the NSW Government's Department of Primary Industries.Ian Paulsen is supported by an Australian Research Council Laureate Fellowship.TCW and MIE are supported by the CSIRO Synthetic Biology Future Science Platform and Macquarie University.
文摘One-carbon compounds,such as methanol,are becoming potential alternatives to sugars as feedstocks for the biological production of chemicals,fuels,foods,and pharmaceuticals.Efficient biological production often requires extensive genetic manipulation of a microbial host strain,making well-characterised and geneticallytractable model organisms like the yeast Saccharomyces cerevisiae attractive targets for the engineering of methylotrophic metabolism.S.cerevisiae strains S288C and CEN.PK are the two best-characterised and most widely used hosts for yeast synthetic biology and metabolic engineering,yet they have unpredictable metabolic phenotypes related to their many genomic differences.We therefore sought to benchmark these two strains as potential hosts for engineered methylotrophic metabolism by comparing their growth and transcriptomic responses to methanol.CEN.PK had improved growth in the presence of methanol relative to the S288C derivative BY4741.The CEN.PK transcriptome also had a specific and relevant response to methanol that was either absent or less pronounced in the BY4741 strain.This response included up-regulation of genes associated with mitochondrial and peroxisomal metabolism,alcohol and formate dehydrogenation,glutathione metabolism,and the global transcriptional regulator of metabolism MIG3.Over-expression of MIG3 enabled improved growth in the presence of methanol,suggesting that MIG3 is a mediator of the superior CEN.PK strain growth.CEN.PK was therefore identified as a superior strain for the future development of synthetic methylotrophy in S.cerevisiae.
