Formate bioconversion plays a crucial role in achieving renewable resource utilization and green and sustainable development,as it helps convert formate to biofuels and biochemicals.However,to tap the full potential o...Formate bioconversion plays a crucial role in achieving renewable resource utilization and green and sustainable development,as it helps convert formate to biofuels and biochemicals.However,to tap the full potential of formate bioconversion,it is important to identify the most appropriate microbial hosts,design the most promising formate assimilation pathways,and develop the most efficient formate assimilation cell factories.Here,we summarize the formatotrophic microorganisms capable of assimilating formate into building blocks of cell growth and analyze the characteristics of formate assimilation pathways for transmitting formate into central carbon metabolism.Furthermore,we discuss microbial engineering strategies to improve the efficiency of formate utilization for producing high-value bioproducts.Finally,we highlight the key challenges of formate bioconversion and their possible solutions to advance the formate bioeconomy and biomanufacturing.展开更多
For the efficient conversion of L-tyrosine(L-Tyr)to tyrosol,which is an aromatic compound widely used in the pharmaceutical and chemical industries,a novel four-enzyme cascade pathway based on the Ehrlich pathway of S...For the efficient conversion of L-tyrosine(L-Tyr)to tyrosol,which is an aromatic compound widely used in the pharmaceutical and chemical industries,a novel four-enzyme cascade pathway based on the Ehrlich pathway of Saccharomyces cerevisiae was designed and reconstructed in Escherichia coli.Then,the expression levels of the relevant enzymes were coordinated using a modular approach and gene duplication after the identification of the pyruvate decarboxylase from Candida tropicalis(CtPDC)as the rate-limiting enzymatic step.In situ product removal(ISPR)strategy with XAD4 resins was explored to avoid product inhibition and further improve tyrosol yield.As a result,the titer and conversion rate of tyrosol obtained were 35.7 g·L^(-1) and 93.6%,respectively,in a 3-L bioreactor.Results presented here provide a potential enzymatic process for industrial production of tyrosol from cheap amino acids.展开更多
L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the p...L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.展开更多
Succinate is an important building block for chemical synthesis.However,during the fermentation process,excessive osmotic stress and byproduct accumulation substantively impair the performance of the microbial cell fa...Succinate is an important building block for chemical synthesis.However,during the fermentation process,excessive osmotic stress and byproduct accumulation substantively impair the performance of the microbial cell factory.To this end,two strategies were proposed.First,an osmo-tolerant mutant,Escherichia coli FMME-N-2,was screened by combined mutagenesis(ARTP and^(60)Co-γirradiation)to produce 51.8 g L^(−1)succinate with a productivity of 0.81 g L^(−1)h^(−1).Second,an oxygen-dependent bifunctional switch(OBS)was developed with promoter PfnrF8-based activation and tobacco etch virus protease-based inhibition functions.With ribosomal binding site(RBS)and degron optimization of OBS,the optimal strain E.coli FMME-N-30 achieved a succinate titer and productivity of 119 g L^(−1)and 1.65 g L^(−1) h^(−1),respectively,in a 30-L fermentor,while only 7.1 g L^(−1)acetate and no formate or lactate were detected.Compared to the wild-type strain E.coli FMME-N,the succinate titer was increased by 3.3-fold.These results highlight the applicability of OBS for the large-scale production of value-added chemicals.展开更多
The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to a...The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals.Thus,the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications.In this review,we provide an in-depth discussion on the properties of natural and synthetic methylotrophs,and summarize the natural and synthetic methanol assimilation pathways.Further,we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals.Finally,we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.展开更多
As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In th...As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In this study,Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method.To improve the production of protease,atmospheric room temperature plasma(ARTP)mutagenesis was used in the first round,protease activity reached 315.0 U/mL.Then,to enhance production of protease,^(60)Co-γ irradiation was used for combined mutagenesis,leading to protease activity of B.amyloliquefaciens FMME ZK003 up to 355.0 U/mL.Furthermore,to realize the efficient production of protease,after optimization of fermentation conditions,protease activity was increased to 456.9 U/mL.Finally,protease activity of B.amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter.These results indicate that B.amyloliquefaciens can efficiently produce protease,which provides a good foundation for the industrial production of protease.展开更多
The mycelial bacterium Streptomyces is a workhorse for producing natural products,serving as a key source of drugs and other valuable chemicals.However,its complicated life cycle,silent biosynthetic gene clusters(BGCs...The mycelial bacterium Streptomyces is a workhorse for producing natural products,serving as a key source of drugs and other valuable chemicals.However,its complicated life cycle,silent biosynthetic gene clusters(BGCs),and poorly characterized metabolic mechanisms limit efficient production of natural products.There-fore,a metabolic engineering strategy,including traditional and emerging tools from different disciplines,was developed to further enhance natural product synthesis by Streptomyces.Here,current trends in systems metabolic engineering,including tools and strategies,are reviewed.Particularly,this review focuses on recent developments in the selection of methods for regulating the Streptomyces life cycle,strategies for the activation of silent gene clusters,and the exploration of regulatory mechanisms governing antibiotic production.Finally,future challenges and prospects are discussed.展开更多
Cytochrome P450 enzymes(CYPs)catalyze a series of C-H and C=C oxygenation reactions,including hydroxylation,epoxidation,and ketonization.They are attractive biocatalysts because of their ability to selectively introdu...Cytochrome P450 enzymes(CYPs)catalyze a series of C-H and C=C oxygenation reactions,including hydroxylation,epoxidation,and ketonization.They are attractive biocatalysts because of their ability to selectively introduce oxygen into inert molecules under mild conditions.This review provides a comprehensive overview of the C-H and C=C oxygenation reactions catalyzed by CYPs and the various strategies for achieving higher selectivity and enzymatic activity.Furthermore,we discuss the application of C-H and C=C oxygenation catalyzed by CYPs to obtain the desired chemicals or pharmaceutical intermediates in practical production.The rapid development of protein engineering for CYPs provides excellent biocatalysts for selective C-H and C=C oxygenation reactions,thereby promoting the development of environmentally friendly and sustainable production processes.展开更多
The use of abundant and cheap one carbon(C1)feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems.The conversion of C1 feedstocks to h...The use of abundant and cheap one carbon(C1)feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems.The conversion of C1 feedstocks to high-value chemicals is dependent on efficient C1 assimilation pathways and microbial chassis adapted for efficient incorporation.Here,we opted to summarize the natural and synthetic C1 assimilation pathways and their key factors for metabolizing C1 feedstock.Accordingly,we discussed the metabolic engineering strategies for enabling the microbial utilization of C1 feedstocks for the bioproduction of value-added chemicals.In addition,we highlighted future perspectives of C1-based biomanufacturing for achieving a low-carbon footprint for the biosynthesis of chemicals.展开更多
Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages,including mild reaction conditions and sustainable manufacturing.However,low-efficiency biorefineries always result ...Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages,including mild reaction conditions and sustainable manufacturing.However,low-efficiency biorefineries always result in an uncompetitive biological process compared to the current petrochemical process.Thus,improving microbial capacity to maximize product yield,productivity,and titer has been recognized as a central goal for bioengineers and biochemists.The knowledge of cellular biochemistry has enabled the regulation of microbial physiology to couple with chemical production.The rapid development in metabolic engineering provides diverse strategies to enhance the efficiency of chemical biosynthesis pathways.New synthetic biology tools as well as novel regulatory targets also offer the opportunity to improve biorefinery environmental adaptivity.In this review,the recent advances in building efficient biorefineries were showcased.In addition,the challenges and future perspectives of microbial host engineering for increased microbial capacity of a biorefinery were discussed.展开更多
D-p-hydroxyphenylglycine(D-HPG)is an important intermediate in the pharmaceutical industry,and it is commonly syn-thesized by cascading D-hydantoinase(DHase)and D-carbamoylase(DCase).In this study,the stability of DCa...D-p-hydroxyphenylglycine(D-HPG)is an important intermediate in the pharmaceutical industry,and it is commonly syn-thesized by cascading D-hydantoinase(DHase)and D-carbamoylase(DCase).In this study,the stability of DCase was identified as the main problem that limits its application.Therefore,the complexed structure of AkDCase(DCase from the Agrobacterium sp.strain KNK712)with the substrate N-carbamoyl-D-p-hydroxyphenylglycine(CpHPG)(with 2.52Åresolution)and catalytic mechanism were resolved.Based on the catalytic mechanism and electrostatic stabilization,salt bridge engineering was adopted to improve AkDCase thermostability.The best variant,AkDCase^(D30A),increased theTm by 2.91℃and half-life(t_(1/2))at 40 and 60℃by 18.43 h and 23.21 min,respectively.After AkDCase^(D30A) was assembled with GsDHase(DHase from Geobacillus stearothermophilus SD-1)in a single Escherichia coli cell,the recombinant strain could produce 29.53 g/L D-HPG within 12 h,with a 97%conversion and a 2.46 g/(L·h)space-time yield(STY).The titer of D-HPG increased by 40.55%compared to the E.coli cell harboring pETduet-1-AkDCase-GsDHase.The recombinant strain could be used for two cycles.Our research provides a basis for the industrial production of D-HPG.展开更多
基金supported by the National Natural Science Foundation of China(22378166)the Basic Research Program of Jiangsu and Jiangsu Basic Research Center for Synthetic Biology(BK20233003)+1 种基金the Fundamental Research Funds for the Central Universities(JUSRP622001)the Open Funding Project of Key Laboratory of Industrial Biotechnology Ministry of Education(KLIB-KF202403).
文摘Formate bioconversion plays a crucial role in achieving renewable resource utilization and green and sustainable development,as it helps convert formate to biofuels and biochemicals.However,to tap the full potential of formate bioconversion,it is important to identify the most appropriate microbial hosts,design the most promising formate assimilation pathways,and develop the most efficient formate assimilation cell factories.Here,we summarize the formatotrophic microorganisms capable of assimilating formate into building blocks of cell growth and analyze the characteristics of formate assimilation pathways for transmitting formate into central carbon metabolism.Furthermore,we discuss microbial engineering strategies to improve the efficiency of formate utilization for producing high-value bioproducts.Finally,we highlight the key challenges of formate bioconversion and their possible solutions to advance the formate bioeconomy and biomanufacturing.
基金financially supported by the Fundamental Research Funds for the Central Universities (JUSRP21915)National Natural Science Foundation of China (22008089, 21878126)+2 种基金Provincial Natural Science Foundation of Jiangsu Province(BK20200622)the key technologies Research&Development Program of Jiangsu Province (BE2018623)the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-20)
文摘For the efficient conversion of L-tyrosine(L-Tyr)to tyrosol,which is an aromatic compound widely used in the pharmaceutical and chemical industries,a novel four-enzyme cascade pathway based on the Ehrlich pathway of Saccharomyces cerevisiae was designed and reconstructed in Escherichia coli.Then,the expression levels of the relevant enzymes were coordinated using a modular approach and gene duplication after the identification of the pyruvate decarboxylase from Candida tropicalis(CtPDC)as the rate-limiting enzymatic step.In situ product removal(ISPR)strategy with XAD4 resins was explored to avoid product inhibition and further improve tyrosol yield.As a result,the titer and conversion rate of tyrosol obtained were 35.7 g·L^(-1) and 93.6%,respectively,in a 3-L bioreactor.Results presented here provide a potential enzymatic process for industrial production of tyrosol from cheap amino acids.
基金financially supported by the national first-class discipline program of Light Industry Technology and Engineering(LITE201820)the Key Technologies Research and Development Program of Jiangsu Province(BE2018623)。
文摘L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.
基金This work was fnancially supported by the National Key R&D Program of China(2020YFA0908500)the Key Program of the National Natural Science Foundation of China(22038005)+1 种基金the Science Fund for Creative Research Groups of the National Science Foundation of China(32021005)the national frst-class discipline program of Light Industry Technology and Engineering(LITE2018-08).
文摘Succinate is an important building block for chemical synthesis.However,during the fermentation process,excessive osmotic stress and byproduct accumulation substantively impair the performance of the microbial cell factory.To this end,two strategies were proposed.First,an osmo-tolerant mutant,Escherichia coli FMME-N-2,was screened by combined mutagenesis(ARTP and^(60)Co-γirradiation)to produce 51.8 g L^(−1)succinate with a productivity of 0.81 g L^(−1)h^(−1).Second,an oxygen-dependent bifunctional switch(OBS)was developed with promoter PfnrF8-based activation and tobacco etch virus protease-based inhibition functions.With ribosomal binding site(RBS)and degron optimization of OBS,the optimal strain E.coli FMME-N-30 achieved a succinate titer and productivity of 119 g L^(−1)and 1.65 g L^(−1) h^(−1),respectively,in a 30-L fermentor,while only 7.1 g L^(−1)acetate and no formate or lactate were detected.Compared to the wild-type strain E.coli FMME-N,the succinate titer was increased by 3.3-fold.These results highlight the applicability of OBS for the large-scale production of value-added chemicals.
基金supported by the National Natural Science Founda-tion of China(22122806 and 22038005)the Major Project of Natu-ral Science Foundation of Jiangsu Province(BK20212013)+1 种基金the Provin-cial Outstanding Youth Foundation of Jiangsu Province(BK20211529)the Fundamental Research Funds for the Central Universities(JUSRP22031).
文摘The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals.Thus,the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications.In this review,we provide an in-depth discussion on the properties of natural and synthetic methylotrophs,and summarize the natural and synthetic methanol assimilation pathways.Further,we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals.Finally,we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.
基金the Provincal Outstanding Youth Foundation of Jiangsu Province(BK20211529)the National Science Fund for Excellent Young Scholars(22122806).
文摘As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In this study,Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method.To improve the production of protease,atmospheric room temperature plasma(ARTP)mutagenesis was used in the first round,protease activity reached 315.0 U/mL.Then,to enhance production of protease,^(60)Co-γ irradiation was used for combined mutagenesis,leading to protease activity of B.amyloliquefaciens FMME ZK003 up to 355.0 U/mL.Furthermore,to realize the efficient production of protease,after optimization of fermentation conditions,protease activity was increased to 456.9 U/mL.Finally,protease activity of B.amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter.These results indicate that B.amyloliquefaciens can efficiently produce protease,which provides a good foundation for the industrial production of protease.
基金supported by the Science Fund for Creative Re-search Groups of the National Natural Science Foundation of China(32021005)and the National Key R&D Program of China(No.2018YFA0901400).
文摘The mycelial bacterium Streptomyces is a workhorse for producing natural products,serving as a key source of drugs and other valuable chemicals.However,its complicated life cycle,silent biosynthetic gene clusters(BGCs),and poorly characterized metabolic mechanisms limit efficient production of natural products.There-fore,a metabolic engineering strategy,including traditional and emerging tools from different disciplines,was developed to further enhance natural product synthesis by Streptomyces.Here,current trends in systems metabolic engineering,including tools and strategies,are reviewed.Particularly,this review focuses on recent developments in the selection of methods for regulating the Streptomyces life cycle,strategies for the activation of silent gene clusters,and the exploration of regulatory mechanisms governing antibiotic production.Finally,future challenges and prospects are discussed.
基金supported by the National Key R&D Program of China(Grant No.2021YFC2100100 and 2021YFC2102000)the General Program of National Natural Science Foundation of China(Grant No.22178146).
文摘Cytochrome P450 enzymes(CYPs)catalyze a series of C-H and C=C oxygenation reactions,including hydroxylation,epoxidation,and ketonization.They are attractive biocatalysts because of their ability to selectively introduce oxygen into inert molecules under mild conditions.This review provides a comprehensive overview of the C-H and C=C oxygenation reactions catalyzed by CYPs and the various strategies for achieving higher selectivity and enzymatic activity.Furthermore,we discuss the application of C-H and C=C oxygenation catalyzed by CYPs to obtain the desired chemicals or pharmaceutical intermediates in practical production.The rapid development of protein engineering for CYPs provides excellent biocatalysts for selective C-H and C=C oxygenation reactions,thereby promoting the development of environmentally friendly and sustainable production processes.
基金supported by the Provincial Outstanding Youth Foundation of Jiangsu Province(BK20211529)the National Science Fund for Excellent Young Scholars(22122806)the Fundamental Research Funds for the Central Universities(JUSRP22031).
文摘The use of abundant and cheap one carbon(C1)feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems.The conversion of C1 feedstocks to high-value chemicals is dependent on efficient C1 assimilation pathways and microbial chassis adapted for efficient incorporation.Here,we opted to summarize the natural and synthetic C1 assimilation pathways and their key factors for metabolizing C1 feedstock.Accordingly,we discussed the metabolic engineering strategies for enabling the microbial utilization of C1 feedstocks for the bioproduction of value-added chemicals.In addition,we highlighted future perspectives of C1-based biomanufacturing for achieving a low-carbon footprint for the biosynthesis of chemicals.
基金supported by the National Key R and D Program of China(2020YFA0908300)the Science Fund for Creative Research Groups of the National Natural Science Foundation of China(32021005)+1 种基金the Key Program of the National Natural Science Foundation of China(22038005)the National Natural Science Foundation of China(32070124,22008087).
文摘Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages,including mild reaction conditions and sustainable manufacturing.However,low-efficiency biorefineries always result in an uncompetitive biological process compared to the current petrochemical process.Thus,improving microbial capacity to maximize product yield,productivity,and titer has been recognized as a central goal for bioengineers and biochemists.The knowledge of cellular biochemistry has enabled the regulation of microbial physiology to couple with chemical production.The rapid development in metabolic engineering provides diverse strategies to enhance the efficiency of chemical biosynthesis pathways.New synthetic biology tools as well as novel regulatory targets also offer the opportunity to improve biorefinery environmental adaptivity.In this review,the recent advances in building efficient biorefineries were showcased.In addition,the challenges and future perspectives of microbial host engineering for increased microbial capacity of a biorefinery were discussed.
基金supported by the National Key R&D Program of China(Grant No.2021YFC2100100)the General Program of National Natural Science Foundation of China(22178146)+1 种基金the Program for Young Talents in Chinathe Fundamental Research Funds for the Central Universities(JUSRP622011).
文摘D-p-hydroxyphenylglycine(D-HPG)is an important intermediate in the pharmaceutical industry,and it is commonly syn-thesized by cascading D-hydantoinase(DHase)and D-carbamoylase(DCase).In this study,the stability of DCase was identified as the main problem that limits its application.Therefore,the complexed structure of AkDCase(DCase from the Agrobacterium sp.strain KNK712)with the substrate N-carbamoyl-D-p-hydroxyphenylglycine(CpHPG)(with 2.52Åresolution)and catalytic mechanism were resolved.Based on the catalytic mechanism and electrostatic stabilization,salt bridge engineering was adopted to improve AkDCase thermostability.The best variant,AkDCase^(D30A),increased theTm by 2.91℃and half-life(t_(1/2))at 40 and 60℃by 18.43 h and 23.21 min,respectively.After AkDCase^(D30A) was assembled with GsDHase(DHase from Geobacillus stearothermophilus SD-1)in a single Escherichia coli cell,the recombinant strain could produce 29.53 g/L D-HPG within 12 h,with a 97%conversion and a 2.46 g/(L·h)space-time yield(STY).The titer of D-HPG increased by 40.55%compared to the E.coli cell harboring pETduet-1-AkDCase-GsDHase.The recombinant strain could be used for two cycles.Our research provides a basis for the industrial production of D-HPG.
