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
Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food,cosmetics,pharmaceutical,and nutraceutical industries.Synthetic biology and metabolic engineering enable mi...Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food,cosmetics,pharmaceutical,and nutraceutical industries.Synthetic biology and metabolic engineering enable microbes,especially the model organism Saccharomyces cerevisiae(generally recognised as safe)to possess great potential for the production of vitamin A.Herein,we first generated a vitamin A-producing strain by miningβ-carotene 15,15′-mono(di)oxygenase from different sources and identified two isoenzymes Mbblh and Ssbco with comparable catalytic properties but different catalytic mechanisms.Combinational expression of isoenzymes increased the flux fromβ-carotene to vitamin A metabolism.To modulate the vitamin A components,retinol dehydrogenase 12 from Homo sapiens was introduced to achieve more than 90%retinol purity using shake flask fermentation.Overexpressing POS5Δ17 enhanced the reduced nicotinamide adenine dinucleotide phosphate pool,and the titer of vitamin A was elevated by almost 46%.Multi-copy integration of the key rate-limiting step gene Mbblh further improved the synthesis of vitamin A.Consequently,the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level.Eventually,the highest reported titer of 5.21 g/L vitamin A in S.cerevisiae was achieved in a 1-L bioreactor.This study unlocked the potential of S.cerevisiae for synthesising vitamin A in a sustainable and economical way,laying the foundation for the commercial-scale production of bio-based vitamin A.展开更多
2′-fucosyllactose(2′-FL)holds significant role in the infants’nutrition.While microbial production of 2′-FL has predominantly utilized Escherichia coli and Saccharomyces cerevisiae,the potential of Pichia pastoris...2′-fucosyllactose(2′-FL)holds significant role in the infants’nutrition.While microbial production of 2′-FL has predominantly utilized Escherichia coli and Saccharomyces cerevisiae,the potential of Pichia pastoris,renowned for its robust NADPH regeneration capability,remains underexplored.Herein,we systematically engineered the metabolism of P.pastoris to develop an efficient 2′-FL-producing cell factory.We first constructed the de novo biosynthesis pathway for 2′-FL in P.pastoris,achieving an initial titer of 0.143 g/L.By optimizing enzyme se-lection and solubility ofα-1,2-fucosyltransferase(FutC),2′-FL production was enhanced by nearly ten folds.Subsequently,engineering NADPH supply further increased the 2′-FL production by 170%.Furthermore,we enhanced energy supply by incorporating an orthogonal energy module based on the methanol dissimilation pathway and increasing GTP availability,resulting in a 32%improvement in 2′-FL production.Finally,through the optimization of fermentation condition,we realized the production titer of 2′-FL to 3.50 g/L in shake-flask,representing the highest titer in P.pastoris.These findings highlight the potential of P.pastoris as a chassis to produce chemicals by providing abundant NADPH and utilizing methanol as co-substrate to supply sufficient energy.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 Research and Development Program(2023YFF1103701)the Key-Area Research and Development Program of Guangdong Province(2020B0303070002)+1 种基金the National Key Research and Development Program of China(2018YFA0900702)the National Natural Science Foundation of China(22178261).
文摘Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food,cosmetics,pharmaceutical,and nutraceutical industries.Synthetic biology and metabolic engineering enable microbes,especially the model organism Saccharomyces cerevisiae(generally recognised as safe)to possess great potential for the production of vitamin A.Herein,we first generated a vitamin A-producing strain by miningβ-carotene 15,15′-mono(di)oxygenase from different sources and identified two isoenzymes Mbblh and Ssbco with comparable catalytic properties but different catalytic mechanisms.Combinational expression of isoenzymes increased the flux fromβ-carotene to vitamin A metabolism.To modulate the vitamin A components,retinol dehydrogenase 12 from Homo sapiens was introduced to achieve more than 90%retinol purity using shake flask fermentation.Overexpressing POS5Δ17 enhanced the reduced nicotinamide adenine dinucleotide phosphate pool,and the titer of vitamin A was elevated by almost 46%.Multi-copy integration of the key rate-limiting step gene Mbblh further improved the synthesis of vitamin A.Consequently,the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level.Eventually,the highest reported titer of 5.21 g/L vitamin A in S.cerevisiae was achieved in a 1-L bioreactor.This study unlocked the potential of S.cerevisiae for synthesising vitamin A in a sustainable and economical way,laying the foundation for the commercial-scale production of bio-based vitamin A.
基金supported by the National Key R&D Program of China(Grant No.2021YFA0911000)the National Natural Science Foundation of China(Grant No.32488301&Grant No.32071416&Grant No.22308369)+2 种基金the Key-Area Research and Development Program of Guangdong Province(Grant No.2022B1111080005)the Strategic Pri-ority Research Program of the Chinese Academy of Sciences(Grant No.XDB0480000)the Shenzhen Science and Technology Major Project(KJZD20240903100207010).
文摘2′-fucosyllactose(2′-FL)holds significant role in the infants’nutrition.While microbial production of 2′-FL has predominantly utilized Escherichia coli and Saccharomyces cerevisiae,the potential of Pichia pastoris,renowned for its robust NADPH regeneration capability,remains underexplored.Herein,we systematically engineered the metabolism of P.pastoris to develop an efficient 2′-FL-producing cell factory.We first constructed the de novo biosynthesis pathway for 2′-FL in P.pastoris,achieving an initial titer of 0.143 g/L.By optimizing enzyme se-lection and solubility ofα-1,2-fucosyltransferase(FutC),2′-FL production was enhanced by nearly ten folds.Subsequently,engineering NADPH supply further increased the 2′-FL production by 170%.Furthermore,we enhanced energy supply by incorporating an orthogonal energy module based on the methanol dissimilation pathway and increasing GTP availability,resulting in a 32%improvement in 2′-FL production.Finally,through the optimization of fermentation condition,we realized the production titer of 2′-FL to 3.50 g/L in shake-flask,representing the highest titer in P.pastoris.These findings highlight the potential of P.pastoris as a chassis to produce chemicals by providing abundant NADPH and utilizing methanol as co-substrate to supply sufficient energy.
