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 ad...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.展开更多
The engineering of microbial cell factories for the production of high-value chemicals from renewable resources presents several challenges,including the optimization of key enzymes,pathway fluxes and metabolic networ...The engineering of microbial cell factories for the production of high-value chemicals from renewable resources presents several challenges,including the optimization of key enzymes,pathway fluxes and metabolic networks.Addressing these challenges involves the development of synthetic auxotrophs,a strategy that links cell growth with enzyme properties or biosynthetic pathways.This linkage allows for the improvement of enzyme properties by in vivo directed enzyme evolution,the enhancement of metabolic pathway fluxes under growth pressure,and remodeling of metabolic networks through directed strain evolution.The advantage of employing synthetic auxotrophs lies in the power of growth-coupled selection,which is not only high-throughput but also labor-saving,greatly simplifying the development of both strains and enzymes.Synthetic auxotrophs play a pivotal role in advancing microbial cell factories,offering benefits from enzyme optimization to the manipulation of metabolic networks within single microbes.Furthermore,this strategy extends to coculture systems,enabling collaboration within microbial communities.This review highlights the recently developed applications of synthetic auxotrophs as microbial cell factories,and discusses future perspectives,aiming to provide a practical guide for growth-coupled models to produce value-added chemicals as part of a sustainable biorefinery.展开更多
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)+3 种基金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).
文摘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.
基金supported by the National Key R&D Program of China(Grant No.2022YFC2106100)the National Natural Science Foundation of China(Grant Nos.22078011,22378016,and 22238001)Guangdong Key Area Research and Development Program(Grant No.2022B1111080003).
文摘The engineering of microbial cell factories for the production of high-value chemicals from renewable resources presents several challenges,including the optimization of key enzymes,pathway fluxes and metabolic networks.Addressing these challenges involves the development of synthetic auxotrophs,a strategy that links cell growth with enzyme properties or biosynthetic pathways.This linkage allows for the improvement of enzyme properties by in vivo directed enzyme evolution,the enhancement of metabolic pathway fluxes under growth pressure,and remodeling of metabolic networks through directed strain evolution.The advantage of employing synthetic auxotrophs lies in the power of growth-coupled selection,which is not only high-throughput but also labor-saving,greatly simplifying the development of both strains and enzymes.Synthetic auxotrophs play a pivotal role in advancing microbial cell factories,offering benefits from enzyme optimization to the manipulation of metabolic networks within single microbes.Furthermore,this strategy extends to coculture systems,enabling collaboration within microbial communities.This review highlights the recently developed applications of synthetic auxotrophs as microbial cell factories,and discusses future perspectives,aiming to provide a practical guide for growth-coupled models to produce value-added chemicals as part of a sustainable biorefinery.
