Vanillin,a valuable aromatic compound,is widely used in the food and medical industries.Vanillin production using cell factory is considered a promising alternative to petro-based synthesis,however,formation of by-pro...Vanillin,a valuable aromatic compound,is widely used in the food and medical industries.Vanillin production using cell factory is considered a promising alternative to petro-based synthesis,however,formation of by-products,insufficient supply of precursors and cofactors have caused the significant challenge to high-level production.In this study,we focused on the de novo synthesis of vanillin in E.coli through preventing the accumulation of by-products,optimizing the supply of L-tyrosine and cofactors,and designing a co-culture system.To reduce vanillic acid accumulation,a carboxylic acid reductase(MmCAR)and its reactivator phos-phopantetheinyl transferase(SFP)was screened,resulting in 88.1%decrease of vanillic acid.Deleting alcohol dehydrogenases(yahK yjgB and yqhD)efficiently inhibited the formation of vanillyl alcohol,resulting in a 4.25-fold increase in vanillin production.Deleting tyrR and pheA,and overexpressing aroG^(D146N )and tyrA^(M53I,A354V) to alleviate their feedback inhibitions further enhanced the titer of vanillin 45.96%.Increasing NADPH regenera-tion by overexpressing pos5(encoding NADH kinase)and promoting S-adenosyl-l-methionine(SAM)regenera-tion by expressing mtn(encoding 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase)and luxS(encoding S-ribosyl-homocysteine lyase)resulted in the titer of vanillin to 279.17 mg/L.Finally,the use of a E.coli co-culture system achieved the production of 465.81 mg/L vanillin,the best reported titer in E.coli.These results provide a new opportunity for de novo production of vanillin from glucose.展开更多
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
D-Tagatose is a functional rare sugar with well-established health benefits and considerable potential for use in the pharmaceutical and food sectors.Although redox-based biosynthesis serves to overcome the thermodyna...D-Tagatose is a functional rare sugar with well-established health benefits and considerable potential for use in the pharmaceutical and food sectors.Although redox-based biosynthesis serves to overcome the thermodynamic equilibrium limitations in D-tagatose production,its reaction efficiency remains a critical bottleneck.Here,we developed an engineered Escherichia coli catalyst for high-efficiency microbial production of D-tagatose.The biosynthetic pathway was first constructed by co-expressing galactitol-2-dehydrogenase(GDH)and xylose reductase(XR)to direct galactose-derived carbon flow toward D-tagatose.Cofactor engineering was then applied to fine-tune the balance between NADPH and NAD+.This adjusted the initial NAD^(+)/NADH and NADP^(+)/NADPH ratios from 0.67 to 3.42 to 0.77 and 0.88,respectively,improving the redox balance and overall performance of the pathway.Systematic metabolic tuning resulted in a 7.23-fold boost in D-tagatose generation,with the final titer reaching 34.92 g/L(yield:0.87 g/g D-galactose,space-time yield:0.55 g/L⋅h)under optimized whole-cell catalytic conditions.We demonstrate that the targeted modulation of NADPH-NAD+homeostasis offers a generalizable strategy for enhancing redox-dependent biotransformations.This approach provides a sustainable alternative for rare sugar biosynthesis.展开更多
6ʹ-Sialyllactose(6′-SL)is the most abundant sialylated oligosaccharide in breast milk,recognized for its beneficial effects on human health,particularly in infant brain development.This study focused on enhancing the...6ʹ-Sialyllactose(6′-SL)is the most abundant sialylated oligosaccharide in breast milk,recognized for its beneficial effects on human health,particularly in infant brain development.This study focused on enhancing the de novo synthesis of 6′-SL in engineered E.coli.A glutamine cycling system was established,and genes encoding collateral pathways in the 6′-SL metabolic pathway were inhibited.To overcome the bottleneck posed by the rate-limiting enzyme α2,6-sialyltransferase(α2,6-siaT),a multi-level combinatorial strategy was employed,including screening for ribosome-binding site(RBS)substitutions,fusion protein creation,and multi-copy gene expression.This approach facilitated the efficient utilization of α2,6-siaT in the 6′-SL biosynthetic pathway.Additionally,coordinated expression of cytidine triphosphate(CTP)cofactor engineering optimized the intracellular supply of the precursor cytidine-5ʹ-monophospho-N-acetylneuraminic acid(CMP-Neu5Ac)and synchronized the synthesis rate of 6′-SL,resulting in the high-yielding strain KA34.Strain KA34 achieved a titer of 3.85 g/L in shake flask cultures and 25.31 g/L in a 3-L bioreactor,with a productivity of 0.36 g/L/h and a lactose conversion yield of 0.55 mol 6ʹ-SL/mol.This study demonstrates a successful strategy for optimizing the microbial production of 6ʹ-SL.展开更多
基金supported by National Natural Science Foundation of China(Grant No.22478255).
文摘Vanillin,a valuable aromatic compound,is widely used in the food and medical industries.Vanillin production using cell factory is considered a promising alternative to petro-based synthesis,however,formation of by-products,insufficient supply of precursors and cofactors have caused the significant challenge to high-level production.In this study,we focused on the de novo synthesis of vanillin in E.coli through preventing the accumulation of by-products,optimizing the supply of L-tyrosine and cofactors,and designing a co-culture system.To reduce vanillic acid accumulation,a carboxylic acid reductase(MmCAR)and its reactivator phos-phopantetheinyl transferase(SFP)was screened,resulting in 88.1%decrease of vanillic acid.Deleting alcohol dehydrogenases(yahK yjgB and yqhD)efficiently inhibited the formation of vanillyl alcohol,resulting in a 4.25-fold increase in vanillin production.Deleting tyrR and pheA,and overexpressing aroG^(D146N )and tyrA^(M53I,A354V) to alleviate their feedback inhibitions further enhanced the titer of vanillin 45.96%.Increasing NADPH regenera-tion by overexpressing pos5(encoding NADH kinase)and promoting S-adenosyl-l-methionine(SAM)regenera-tion by expressing mtn(encoding 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase)and luxS(encoding S-ribosyl-homocysteine lyase)resulted in the titer of vanillin to 279.17 mg/L.Finally,the use of a E.coli co-culture system achieved the production of 465.81 mg/L vanillin,the best reported titer in E.coli.These results provide a new opportunity for de novo production of vanillin from glucose.
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.
基金funded by the National Key Research and Development Program of China(2024YFD2200804-4)the Natural Science Foundation of Guangxi Province(2025GXNSFAA069706)the Innovation Project of Guangxi Graduate Education(YCSW2025127).
文摘D-Tagatose is a functional rare sugar with well-established health benefits and considerable potential for use in the pharmaceutical and food sectors.Although redox-based biosynthesis serves to overcome the thermodynamic equilibrium limitations in D-tagatose production,its reaction efficiency remains a critical bottleneck.Here,we developed an engineered Escherichia coli catalyst for high-efficiency microbial production of D-tagatose.The biosynthetic pathway was first constructed by co-expressing galactitol-2-dehydrogenase(GDH)and xylose reductase(XR)to direct galactose-derived carbon flow toward D-tagatose.Cofactor engineering was then applied to fine-tune the balance between NADPH and NAD+.This adjusted the initial NAD^(+)/NADH and NADP^(+)/NADPH ratios from 0.67 to 3.42 to 0.77 and 0.88,respectively,improving the redox balance and overall performance of the pathway.Systematic metabolic tuning resulted in a 7.23-fold boost in D-tagatose generation,with the final titer reaching 34.92 g/L(yield:0.87 g/g D-galactose,space-time yield:0.55 g/L⋅h)under optimized whole-cell catalytic conditions.We demonstrate that the targeted modulation of NADPH-NAD+homeostasis offers a generalizable strategy for enhancing redox-dependent biotransformations.This approach provides a sustainable alternative for rare sugar biosynthesis.
基金financially supported by the Key R&D Program of Shandong Province,China(2024CXGC010615)the China Post-doctoral Science Foundation(2024M751156).
文摘6ʹ-Sialyllactose(6′-SL)is the most abundant sialylated oligosaccharide in breast milk,recognized for its beneficial effects on human health,particularly in infant brain development.This study focused on enhancing the de novo synthesis of 6′-SL in engineered E.coli.A glutamine cycling system was established,and genes encoding collateral pathways in the 6′-SL metabolic pathway were inhibited.To overcome the bottleneck posed by the rate-limiting enzyme α2,6-sialyltransferase(α2,6-siaT),a multi-level combinatorial strategy was employed,including screening for ribosome-binding site(RBS)substitutions,fusion protein creation,and multi-copy gene expression.This approach facilitated the efficient utilization of α2,6-siaT in the 6′-SL biosynthetic pathway.Additionally,coordinated expression of cytidine triphosphate(CTP)cofactor engineering optimized the intracellular supply of the precursor cytidine-5ʹ-monophospho-N-acetylneuraminic acid(CMP-Neu5Ac)and synchronized the synthesis rate of 6′-SL,resulting in the high-yielding strain KA34.Strain KA34 achieved a titer of 3.85 g/L in shake flask cultures and 25.31 g/L in a 3-L bioreactor,with a productivity of 0.36 g/L/h and a lactose conversion yield of 0.55 mol 6ʹ-SL/mol.This study demonstrates a successful strategy for optimizing the microbial production of 6ʹ-SL.
