Terpenoids,known for their structural and functional diversity,are highly valued,especially in food,cosmetics,and cleaning products.Microbial biosynthesis has emerged as a sustainable and environmentally friendly appr...Terpenoids,known for their structural and functional diversity,are highly valued,especially in food,cosmetics,and cleaning products.Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids.However,the natural enzymes involved in the synthesis of terpenoids have problems such as low activity,poor specificity,and insufficient stability,which limit the biosynthesis efficiency.Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids.By modifying the structures and functions of key enzymes,researchers have significantly improved the catalytic activity,specificity,and stability of enzymes related to terpenoid synthesis,providing strong support for the sustainable production of terpenoids.This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids,including improving enzyme activity and stability,changing specificity,and promoting mass transfer through multi-enzyme collaboration.Additionally,this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.展开更多
With the gradual rise of enzyme engineering,it has played an essential role in synthetic biology,medicine,and biomanufacturing.However,due to the limitation of the cell membrane,the complexity of cellular metabolism,t...With the gradual rise of enzyme engineering,it has played an essential role in synthetic biology,medicine,and biomanufacturing.However,due to the limitation of the cell membrane,the complexity of cellular metabolism,the difficulty of controlling the reaction environment,and the toxicity of some metabolic products in traditional in vivo enzyme engineering,it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds.Recently,cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies.This cell-free method has no limitation of the cell membrane and no need to maintain cell viability,and each biosynthetic pathway is highly flexible.This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize.This article aims to discuss the latest advances in cell-free enzyme engineering,assess the trend of this developing topical filed,and analyze its prospects.展开更多
Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmac...Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmaceutical field.The application of enzymes in medicine is diverse,such as:diagnosis,prevention and treatment of diseases with enzymes,manufacture of various drugs with enzymes,etc.,mainly through manual operations,to obtain enzymes required by the pharmaceutical industry,and through various means Enzymes perform their catalytic functions.This article mainly introduces the application of enzyme engineering in the pharmaceutical field,and also prospects the development trend of enzyme engineering in the pharmaceutical field.展开更多
The trend of employing machine learning methods has been increasing to develop promising biocatalysts.Leveraging the experimental findings and simulation data,these methods facilitate enzyme engineering and even the d...The trend of employing machine learning methods has been increasing to develop promising biocatalysts.Leveraging the experimental findings and simulation data,these methods facilitate enzyme engineering and even the design of new-to-nature enzymes.This review focuses on the application of machine learning methods in the engineering of polyethylene terephthalate(PET)hydrolases,enzymes that have the potential to help address plastic pollution.We introduce an overview of machine learning workflows,useful methods and tools for protein design and engineering,and discuss the recent progress of machine learning-aided PET hydrolase engineering and de novo design of PET hydrolases.Finally,as machine learning in enzyme engineering is still evolving,we foresee that advancements in computational power and quality data resources will considerably increase the use of data-driven approaches in enzyme engineering in the coming decades.展开更多
This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter des...This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.展开更多
Graphical abstract 1.Multidisciplinary strategies to enhance therapeutic effects of flavonoids from Epimedi Folium:Integration of herbal medicine,enzyme engineering,and nanotechnology Yi Lu,QiulanLuo,Xiaobin Jia,James...Graphical abstract 1.Multidisciplinary strategies to enhance therapeutic effects of flavonoids from Epimedi Folium:Integration of herbal medicine,enzyme engineering,and nanotechnology Yi Lu,QiulanLuo,Xiaobin Jia,James P.Tam,Huan Yang.展开更多
Biosynthesis—a process utilizing biological systems to synthesize chemical compounds—has emerged as a revolutionary solution to 21st-century challenges due to its environmental sustainability,scalability,and high st...Biosynthesis—a process utilizing biological systems to synthesize chemical compounds—has emerged as a revolutionary solution to 21st-century challenges due to its environmental sustainability,scalability,and high stereoselectivity and regioselectivity.Recent advancements in artificial intelligence(AI)are accelerating biosynthesis by enabling intelligent design,construction,and optimization of enzymatic reactions and biological systems.We first introduce the molecular retrosynthesis route planning in biochemical pathway design,including single-step retrosynthesis algorithms and AI-based chemical retrosynthesis route design tools.We highlight the advantages and challenges of large language models in addressing the sparsity of chemical data.Furthermore,we review enzyme discovery methods based on sequence and structure alignment techniques.Breakthroughs in AI-based structural prediction methods are expected to significantly improve the accuracy of enzyme discovery.We also summarize methods for de novo enzyme generation for nonnatural or orphan reactions,focusing on AI-based enzyme functional annotation and enzyme discovery techniques based on reaction or small molecule similarity.Turning to enzyme engineering,we discuss strategies to improve enzyme thermostability,solubility,and activity,as well as the applications of AI in these fields.The shift from traditional experiment-driven models to data-driven and computationally driven intelligent models is already underway.Finally,we present potential challenges and provide a perspective on future research directions.We envision expanded applications of biocatalysis in drug development,green chemistry,and complex molecule synthesis.展开更多
Plant natural products are a kind of active substance widely used in pharmaceuticals and foods.However,the current production mode based on plant culture and extraction suffer complex processes and severe concerns for...Plant natural products are a kind of active substance widely used in pharmaceuticals and foods.However,the current production mode based on plant culture and extraction suffer complex processes and severe concerns for environmental and ecological.With the increasing awareness of environmental sustainability,engineered microbial cell factories have been an alternative approach to produce natural products.Many engineering strategies have been utilized in microbial biosynthesis of complex phytochemicals such as dynamic control and substructure engineering.Meanwhile,Enzyme engineering including directed evolution and rational design has been implemented to improve enzyme catalysis efficiency and stability as well as change promiscuity to expand product spectra.In this review,we discussed recent advances in microbial biosynthesis of complex phytochemicals from the following aspects,including pathway construction,strain engineering to boost the production.展开更多
The widespread use of polymers has made our lives increasingly convenient by offering a more convenient and dependable material.However,the challenge of efficiently decomposing these materials has resulted in a surge ...The widespread use of polymers has made our lives increasingly convenient by offering a more convenient and dependable material.However,the challenge of efficiently decomposing these materials has resulted in a surge of polymer waste,posing environment and health risk.Currently,landfill and incineration treatment approaches have notable shortcomings,prompting a shift towards more eco-friendly and sustainable biodegradation approaches.Biodegradation primarily relies on microorganisms,with research focusing on both solitary bacterial strain and multi-strain communities for polymer biodegradation.Furthermore,directed evolution and rational design of enzyme have significantly contributed to the polymer biodegradation process.However,previous reviews often undervaluing the role of multi-strain communities.In this review,we assess the current state of these three significant fields of research,provide practical solutions to issues with polymer biodegradation,and outline potential future directions for the subject.Ultimately,biodegradation,whether facilitated by single bacteria,multi-strain communities,or engineered enzymes,now represents the most effective method for managing waste polymers.展开更多
The synthesis of hydroxy fatty acids(HFAs) from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years. Given that selective asymmetric hydration of non-activated C_C...The synthesis of hydroxy fatty acids(HFAs) from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years. Given that selective asymmetric hydration of non-activated C_C bonds has been proven difficult to achieve with chemical catalysts, enzymatic catalysis by fatty acid hydratases(FAHs) presents an attractive alternative approach to produce value-added HFAs with high regio-, enantioand stereospecificity, as well as excellent atom economy. Even though FAHs have just been investigated as a potential biocatalyst for a decade, remarkable information about FAHs in different aspects is available;however, a comprehensive review has not been archived. Herein, we summarize the research progresses on biochemical characterization, structural and mechanistic determination, enzyme engineering, as well as biotechnological application of FAHs. The current challenges and opportunities for an efficient utilization of FAHs in organic synthesis and industrial applications are critically discussed.展开更多
Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a pivotal enzyme that mediates the fixation of CO_(2).As the most abundant protein on earth,Rubisco has a significant impact on global carbon,water,and nitrog...Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a pivotal enzyme that mediates the fixation of CO_(2).As the most abundant protein on earth,Rubisco has a significant impact on global carbon,water,and nitrogen cycles.However,the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency.This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco,which has been challenging due to its high conservation and limitations in chloroplast transformation techniques.However,recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms,including plant Rubisco,in microorganisms.This breakthrough facilitates the acquisition and evaluation of modified proteins,streamlining the measurement of their activity.Moreover,the establishment of a screening system in E.coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution.Finally,this review emphasizes the utilization of Rubisco in microorganisms,not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes.展开更多
Lignin represents the most abundant renewable aromatic source,while flavonoids are aromatic natural compounds with various health-promoting properties and superior biological activities.The bioconversion of lignin der...Lignin represents the most abundant renewable aromatic source,while flavonoids are aromatic natural compounds with various health-promoting properties and superior biological activities.The bioconversion of lignin derivatives into flavonoids holds promising potential for both lignin valorization and flavonoid synthesis.In this review,we prospect sustainable,atom-economic functionalization routes from lignin-derived aromatics to flavonoids by leveraging lignin’s inherent aromaticity.The representative flavonoid biosynthesis routes had first been elaborated from lignin derivatives in detail.The functionalization reactions involved in incorporating lignin derivatives into flavonoid structure were summarized to promote lignin bioconversion and yield flavonoids with desirable properties.Harnessing the powerful engineering strategies,such as synthetic biology,machine learning,metabolic regulation,boost the flavonoid production in microbial cell factories,enhancing lignin valorization.Overall,lignin functionalization routes for flavonoid biosynthesis hold promise to achieve the feasibility of lignin valorization and the production of flavonoids,contributing significantly to the sustainable bioeconomy.展开更多
(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus,which can be used as a raw material in cosmetics and has anti-inflammatory function.In this study,we designed six mutation sites ...(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus,which can be used as a raw material in cosmetics and has anti-inflammatory function.In this study,we designed six mutation sites of the(-)-α-bisabolol synthase BOS using the plmDCA algorithm.Among these,the F324Y mutation demonstrated exceptional performance,increasing the product yield by 73%.We constructed a de novo(-)-α-bisabolol biosynthesis pathways through systematic synthetic biology strategies,including the enzyme design of BOS,selection of different linkers in fusion expression,and optimization of the mevalonate pathway,weakening the branching metabolic flow and multi-copy strategies,the yield of(-)-α-bisabolol was significantly increased,which was nearly 35-fold higher than that of the original strain(2.03 mg/L).The engineered strain was capable of producing 69.7 mg/L in shake flasks.To the best of our knowledge,this is the first report on the biosynthesis of(-)-α-bisabolol in Komagataella phaffii,implying this is a robust cell factory for sustainable production of other terpenoids.展开更多
Terminal deoxynucleotidyl transferase(TdT),a unique DNA polymerase,can elongate DNA by adding deoxynucleotides to the 3′terminal of a DNA chain in a template-independent manner.Owing to their remarkable DNA synthesis...Terminal deoxynucleotidyl transferase(TdT),a unique DNA polymerase,can elongate DNA by adding deoxynucleotides to the 3′terminal of a DNA chain in a template-independent manner.Owing to their remarkable DNA synthesis activity,TdTs have promoted the development of numerous nucleic acid-based methods,tools,and associated applications,attracting broad interest from both academia and industry.This review summarizes and discusses the recent research on TdTs,including their biochemical characteristics,enzyme engineering,and practical applications.New insights and perspectives on the future development of TdTs are provided.展开更多
Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmo- spheric carbon dioxide by photosynthesis. Genetic manip- ulation has increased the variety of chemicals that cyanobact...Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmo- spheric carbon dioxide by photosynthesis. Genetic manip- ulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches： （1） utilization of NADPH- dependent enzymes, （2） increasing NADH production, and （3） changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.展开更多
The aim of this work was to study the efficiency of native lignocellulolytic enzymes obtained from isolated bacteria towards enhanced bioethanol production from lignocellulosic biomass.Maximum cellulose(199.33±0....The aim of this work was to study the efficiency of native lignocellulolytic enzymes obtained from isolated bacteria towards enhanced bioethanol production from lignocellulosic biomass.Maximum cellulose(199.33±0.2 mg/g)and hemicellulose(62.21±0.22 mg/g)content was measured from rice straw in alkali condition compared to acid and biological pretreatment,while significant lignin removal has been observed in biological pretreatment.Saccharification of rice straw using isolated cellulase–xylanase enzymes exhibited 60.33%production of total reducing sugar obtained by commercial cellulase–xylanase cocktail.Maximum glucose,xylose,and total reducing sugar yield of 309±0.32,190.7±0.42,and 499.7±0.37 mg/g,respec-tively,at 37.5℃,pH-7,rice straw concentration of 2.5 g/100 mL,enzyme loading 175μl,and incubation period 42 h by com-mercial cellulase–xylanase enzyme mediated hydrolysis.While in case of using the native cellulase–xylanase cocktail from the isolated bacterial strains,highest yields of glucose,xylose and total reducing sugar production was 253.52±0.56 mg/g,47.94±0.78 mg/g,and 301.46±0.67 mg/g,respectively.While applying the isolated enzymes on alkali-pretreated rice straw,bioethanol concentration of around 32.57±0.25 g/L was recorded after the simultaneous saccharification and fermentation by Saccharomyces cerevisiae.The above mentioned bioethanol concentration was obtained at a process parameter of temperature 35℃,incubation time 58 h,and pH 5.5 for isolated cellulase–xylanase enzymes.A maximum bioethanol concentration using isolated cellulase–xylanase enzymes was nearly 93.89%of bioethanol concentration(34.69±0.28 g/L)obtained using commercial cellulase–xylanase.The present study interpreted that the cutting-edge approach for the native enzymes along with metabolic engineering of the isolated bacteria could be promising towards enhanced bioethanol production.展开更多
Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology.Anthocyanins,the water-soluble color...Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology.Anthocyanins,the water-soluble colored pigments found in terrestrial plants that are responsible for the red,blue and purple coloration of many flowers and fruits,are extensively used in food and cosmetics industry;however,their current supply heavily relies on complex extraction from plant-based materials.A promising alternative is their sustainable production in metabolically engineered microbes.Here,we review the recent progress on anthocyanin biosynthesis in engineered bacteria,with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes,engineering of transportation,regulation of UDP-glucose supply,as well as process optimization.These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.展开更多
Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76...Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.展开更多
Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that ...Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that form active homo-oligomers.From a mechanistic as well as a structural point of view,they are similar to glycoside hydrolases or glycosyltransferases.As the majority of DSPs show strict stereo-and regiospecificities,these enzymes were used to synthesize specific disaccharides.Currently,protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes,to eventually generate a toolbox of new catalysts for glycoside synthesis.Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.展开更多
High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and comme...High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and commercial production of glucose isomerase have paved the way to explore more efficient variants of these enzymes.The 5-hydroxymethylfurfural can be produced from high fructose corn syrup catalytic dehydration,and it can be further converted into various furanic compounds chemically or biologically for various industrial applications as a promising platform chemical.Although the chemical conversion of 5-hydroxymethylfurfural into furanic compounds has been extensively investigated in recent years,bioconversion has shown promise for its mild conditions due to the harsh chemical reaction conditions.This review discusses pro-tein engineering potential for improving glucose isomerase production and recent advancements in bioconversion of 5-hydroxymethylfurfural into value-added furanic derivatives.It suggests bi-ological strategies for the industrial transformation of 5-hydroxymethylfurfural.展开更多
文摘Terpenoids,known for their structural and functional diversity,are highly valued,especially in food,cosmetics,and cleaning products.Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids.However,the natural enzymes involved in the synthesis of terpenoids have problems such as low activity,poor specificity,and insufficient stability,which limit the biosynthesis efficiency.Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids.By modifying the structures and functions of key enzymes,researchers have significantly improved the catalytic activity,specificity,and stability of enzymes related to terpenoid synthesis,providing strong support for the sustainable production of terpenoids.This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids,including improving enzyme activity and stability,changing specificity,and promoting mass transfer through multi-enzyme collaboration.Additionally,this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.
基金supported by the National Key R&D Program of China(2018YFA0901700)the National Natural Science Foundation of China(Nos.21878173,21706144)the Natural Science Foundation of Beijing City(2192023)。
文摘With the gradual rise of enzyme engineering,it has played an essential role in synthetic biology,medicine,and biomanufacturing.However,due to the limitation of the cell membrane,the complexity of cellular metabolism,the difficulty of controlling the reaction environment,and the toxicity of some metabolic products in traditional in vivo enzyme engineering,it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds.Recently,cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies.This cell-free method has no limitation of the cell membrane and no need to maintain cell viability,and each biosynthetic pathway is highly flexible.This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize.This article aims to discuss the latest advances in cell-free enzyme engineering,assess the trend of this developing topical filed,and analyze its prospects.
文摘Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmaceutical field.The application of enzymes in medicine is diverse,such as:diagnosis,prevention and treatment of diseases with enzymes,manufacture of various drugs with enzymes,etc.,mainly through manual operations,to obtain enzymes required by the pharmaceutical industry,and through various means Enzymes perform their catalytic functions.This article mainly introduces the application of enzyme engineering in the pharmaceutical field,and also prospects the development trend of enzyme engineering in the pharmaceutical field.
基金This work was supported by the National Natural Science Foundation of China under grant number 32371325the Seed Funding of China Petrochemical Corporation(Sinopec Group)under grant number 223260the Fundamental Research Funds for the Central Universities(QNTD2023-01).
文摘The trend of employing machine learning methods has been increasing to develop promising biocatalysts.Leveraging the experimental findings and simulation data,these methods facilitate enzyme engineering and even the design of new-to-nature enzymes.This review focuses on the application of machine learning methods in the engineering of polyethylene terephthalate(PET)hydrolases,enzymes that have the potential to help address plastic pollution.We introduce an overview of machine learning workflows,useful methods and tools for protein design and engineering,and discuss the recent progress of machine learning-aided PET hydrolase engineering and de novo design of PET hydrolases.Finally,as machine learning in enzyme engineering is still evolving,we foresee that advancements in computational power and quality data resources will considerably increase the use of data-driven approaches in enzyme engineering in the coming decades.
文摘This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.
文摘Graphical abstract 1.Multidisciplinary strategies to enhance therapeutic effects of flavonoids from Epimedi Folium:Integration of herbal medicine,enzyme engineering,and nanotechnology Yi Lu,QiulanLuo,Xiaobin Jia,James P.Tam,Huan Yang.
基金supported by the National Key R&D Program of China(2023YFA0915500).
文摘Biosynthesis—a process utilizing biological systems to synthesize chemical compounds—has emerged as a revolutionary solution to 21st-century challenges due to its environmental sustainability,scalability,and high stereoselectivity and regioselectivity.Recent advancements in artificial intelligence(AI)are accelerating biosynthesis by enabling intelligent design,construction,and optimization of enzymatic reactions and biological systems.We first introduce the molecular retrosynthesis route planning in biochemical pathway design,including single-step retrosynthesis algorithms and AI-based chemical retrosynthesis route design tools.We highlight the advantages and challenges of large language models in addressing the sparsity of chemical data.Furthermore,we review enzyme discovery methods based on sequence and structure alignment techniques.Breakthroughs in AI-based structural prediction methods are expected to significantly improve the accuracy of enzyme discovery.We also summarize methods for de novo enzyme generation for nonnatural or orphan reactions,focusing on AI-based enzyme functional annotation and enzyme discovery techniques based on reaction or small molecule similarity.Turning to enzyme engineering,we discuss strategies to improve enzyme thermostability,solubility,and activity,as well as the applications of AI in these fields.The shift from traditional experiment-driven models to data-driven and computationally driven intelligent models is already underway.Finally,we present potential challenges and provide a perspective on future research directions.We envision expanded applications of biocatalysis in drug development,green chemistry,and complex molecule synthesis.
基金This work was supported by the National Key Research and Development Program of China(2018YFA0901800)the National Natural Science Foundation of China(No.21736002).
文摘Plant natural products are a kind of active substance widely used in pharmaceuticals and foods.However,the current production mode based on plant culture and extraction suffer complex processes and severe concerns for environmental and ecological.With the increasing awareness of environmental sustainability,engineered microbial cell factories have been an alternative approach to produce natural products.Many engineering strategies have been utilized in microbial biosynthesis of complex phytochemicals such as dynamic control and substructure engineering.Meanwhile,Enzyme engineering including directed evolution and rational design has been implemented to improve enzyme catalysis efficiency and stability as well as change promiscuity to expand product spectra.In this review,we discussed recent advances in microbial biosynthesis of complex phytochemicals from the following aspects,including pathway construction,strain engineering to boost the production.
基金supported by the National Natural Science Foundation of China(22078011,21908003,and 32022002)the National Key Research and Development Program of China(2021YFC2100600)+1 种基金the seed funding of China Petrochemical Corporation(Sinopec Group)(223260)the Fundamental Research Funds for the Central Universities(QNTD2023-01).
文摘The widespread use of polymers has made our lives increasingly convenient by offering a more convenient and dependable material.However,the challenge of efficiently decomposing these materials has resulted in a surge of polymer waste,posing environment and health risk.Currently,landfill and incineration treatment approaches have notable shortcomings,prompting a shift towards more eco-friendly and sustainable biodegradation approaches.Biodegradation primarily relies on microorganisms,with research focusing on both solitary bacterial strain and multi-strain communities for polymer biodegradation.Furthermore,directed evolution and rational design of enzyme have significantly contributed to the polymer biodegradation process.However,previous reviews often undervaluing the role of multi-strain communities.In this review,we assess the current state of these three significant fields of research,provide practical solutions to issues with polymer biodegradation,and outline potential future directions for the subject.Ultimately,biodegradation,whether facilitated by single bacteria,multi-strain communities,or engineered enzymes,now represents the most effective method for managing waste polymers.
基金Financial Supports from Novo Nordisk Foundation(NNF16OC0021740)Aarhus Universitets Forskningsfond,AUFFNOVA(AUFF-E-2015-FLS-9-12)。
文摘The synthesis of hydroxy fatty acids(HFAs) from renewable oil feedstock by addition of water onto C_C bonds has attracted great attention in recent years. Given that selective asymmetric hydration of non-activated C_C bonds has been proven difficult to achieve with chemical catalysts, enzymatic catalysis by fatty acid hydratases(FAHs) presents an attractive alternative approach to produce value-added HFAs with high regio-, enantioand stereospecificity, as well as excellent atom economy. Even though FAHs have just been investigated as a potential biocatalyst for a decade, remarkable information about FAHs in different aspects is available;however, a comprehensive review has not been archived. Herein, we summarize the research progresses on biochemical characterization, structural and mechanistic determination, enzyme engineering, as well as biotechnological application of FAHs. The current challenges and opportunities for an efficient utilization of FAHs in organic synthesis and industrial applications are critically discussed.
基金supported by the National Key R&D Program of China(2021YFC2103500)the National Natural Science Foundation of China(32270057)the DNL Cooperation Fund,CAS(DNL202014).
文摘Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a pivotal enzyme that mediates the fixation of CO_(2).As the most abundant protein on earth,Rubisco has a significant impact on global carbon,water,and nitrogen cycles.However,the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency.This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco,which has been challenging due to its high conservation and limitations in chloroplast transformation techniques.However,recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms,including plant Rubisco,in microorganisms.This breakthrough facilitates the acquisition and evaluation of modified proteins,streamlining the measurement of their activity.Moreover,the establishment of a screening system in E.coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution.Finally,this review emphasizes the utilization of Rubisco in microorganisms,not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes.
基金supported by the National Key Research and Development Program of China(2023YFC3403500)the Key Research and Development Program of Ningxia Hui Autonomous Region(2024BEE02005).
文摘Lignin represents the most abundant renewable aromatic source,while flavonoids are aromatic natural compounds with various health-promoting properties and superior biological activities.The bioconversion of lignin derivatives into flavonoids holds promising potential for both lignin valorization and flavonoid synthesis.In this review,we prospect sustainable,atom-economic functionalization routes from lignin-derived aromatics to flavonoids by leveraging lignin’s inherent aromaticity.The representative flavonoid biosynthesis routes had first been elaborated from lignin derivatives in detail.The functionalization reactions involved in incorporating lignin derivatives into flavonoid structure were summarized to promote lignin bioconversion and yield flavonoids with desirable properties.Harnessing the powerful engineering strategies,such as synthetic biology,machine learning,metabolic regulation,boost the flavonoid production in microbial cell factories,enhancing lignin valorization.Overall,lignin functionalization routes for flavonoid biosynthesis hold promise to achieve the feasibility of lignin valorization and the production of flavonoids,contributing significantly to the sustainable bioeconomy.
基金supported by the key scientific research and development project of Zhejiang Province(2024SSYS0103)the Start-up funds of Xianghu Laboratory(2023C4S02002)the National Natural Science Foundation of China(32301255)。
文摘(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus,which can be used as a raw material in cosmetics and has anti-inflammatory function.In this study,we designed six mutation sites of the(-)-α-bisabolol synthase BOS using the plmDCA algorithm.Among these,the F324Y mutation demonstrated exceptional performance,increasing the product yield by 73%.We constructed a de novo(-)-α-bisabolol biosynthesis pathways through systematic synthetic biology strategies,including the enzyme design of BOS,selection of different linkers in fusion expression,and optimization of the mevalonate pathway,weakening the branching metabolic flow and multi-copy strategies,the yield of(-)-α-bisabolol was significantly increased,which was nearly 35-fold higher than that of the original strain(2.03 mg/L).The engineered strain was capable of producing 69.7 mg/L in shake flasks.To the best of our knowledge,this is the first report on the biosynthesis of(-)-α-bisabolol in Komagataella phaffii,implying this is a robust cell factory for sustainable production of other terpenoids.
基金supported by the National Key Research and Development Program of China(2021YFA0911500)Shandong Provincial Natural Science Foundation(ZR2020ZD23)+1 种基金National Natural Science Foundation of China(32370032,32170088)Taishan Young Scholars(tsqn202312032).
文摘Terminal deoxynucleotidyl transferase(TdT),a unique DNA polymerase,can elongate DNA by adding deoxynucleotides to the 3′terminal of a DNA chain in a template-independent manner.Owing to their remarkable DNA synthesis activity,TdTs have promoted the development of numerous nucleic acid-based methods,tools,and associated applications,attracting broad interest from both academia and industry.This review summarizes and discusses the recent research on TdTs,including their biochemical characteristics,enzyme engineering,and practical applications.New insights and perspectives on the future development of TdTs are provided.
文摘Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmo- spheric carbon dioxide by photosynthesis. Genetic manip- ulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches： （1） utilization of NADPH- dependent enzymes, （2） increasing NADH production, and （3） changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.
文摘The aim of this work was to study the efficiency of native lignocellulolytic enzymes obtained from isolated bacteria towards enhanced bioethanol production from lignocellulosic biomass.Maximum cellulose(199.33±0.2 mg/g)and hemicellulose(62.21±0.22 mg/g)content was measured from rice straw in alkali condition compared to acid and biological pretreatment,while significant lignin removal has been observed in biological pretreatment.Saccharification of rice straw using isolated cellulase–xylanase enzymes exhibited 60.33%production of total reducing sugar obtained by commercial cellulase–xylanase cocktail.Maximum glucose,xylose,and total reducing sugar yield of 309±0.32,190.7±0.42,and 499.7±0.37 mg/g,respec-tively,at 37.5℃,pH-7,rice straw concentration of 2.5 g/100 mL,enzyme loading 175μl,and incubation period 42 h by com-mercial cellulase–xylanase enzyme mediated hydrolysis.While in case of using the native cellulase–xylanase cocktail from the isolated bacterial strains,highest yields of glucose,xylose and total reducing sugar production was 253.52±0.56 mg/g,47.94±0.78 mg/g,and 301.46±0.67 mg/g,respectively.While applying the isolated enzymes on alkali-pretreated rice straw,bioethanol concentration of around 32.57±0.25 g/L was recorded after the simultaneous saccharification and fermentation by Saccharomyces cerevisiae.The above mentioned bioethanol concentration was obtained at a process parameter of temperature 35℃,incubation time 58 h,and pH 5.5 for isolated cellulase–xylanase enzymes.A maximum bioethanol concentration using isolated cellulase–xylanase enzymes was nearly 93.89%of bioethanol concentration(34.69±0.28 g/L)obtained using commercial cellulase–xylanase.The present study interpreted that the cutting-edge approach for the native enzymes along with metabolic engineering of the isolated bacteria could be promising towards enhanced bioethanol production.
基金Funding for some of the work reviewed in this manuscript was provided by the National Science Foundation grant number IIP-1549767.
文摘Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology.Anthocyanins,the water-soluble colored pigments found in terrestrial plants that are responsible for the red,blue and purple coloration of many flowers and fruits,are extensively used in food and cosmetics industry;however,their current supply heavily relies on complex extraction from plant-based materials.A promising alternative is their sustainable production in metabolically engineered microbes.Here,we review the recent progress on anthocyanin biosynthesis in engineered bacteria,with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes,engineering of transportation,regulation of UDP-glucose supply,as well as process optimization.These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.
基金This work was financially supported by the National Key R&D Program of China(2018YFA0900600)the Strategic Priority Research Program "Molecular Mechanism of Plant Growth and Development" of CAS(XDB27020202,XDB27020103)+5 种基金the National Natural Science Foundation of China(31700263,31670099,31700261)grants from the Shanghai Science and Technology Commission(19XD1424500)J.L.is supported by the Foundation of Youth Innovation Promotion Association of the Chinese Academy of SciencesThis work was also financially supported by the Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Science(no.ZSYS-016)the International Partnership Program of Chinese Academy of Science(no.153D31KYSB20170121)the National Key Laboratory of Plant Molecular Genetics,SIPPI,CAS.
文摘Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.
基金This work was supported by the Key Research Program of the Chinese Academy of Sciences(Grant No.ZDRW-ZS-2016-3)the National Natural Science Foundation of China(Grant No.21778073)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(Grant No.TSBICIP-KJGG-003).
文摘Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that form active homo-oligomers.From a mechanistic as well as a structural point of view,they are similar to glycoside hydrolases or glycosyltransferases.As the majority of DSPs show strict stereo-and regiospecificities,these enzymes were used to synthesize specific disaccharides.Currently,protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes,to eventually generate a toolbox of new catalysts for glycoside synthesis.Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.
基金supported by the Natural Sciences and Engineering Research Council of Canada(Grant number RGPIN-2017-05366)to WQ.
文摘High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and commercial production of glucose isomerase have paved the way to explore more efficient variants of these enzymes.The 5-hydroxymethylfurfural can be produced from high fructose corn syrup catalytic dehydration,and it can be further converted into various furanic compounds chemically or biologically for various industrial applications as a promising platform chemical.Although the chemical conversion of 5-hydroxymethylfurfural into furanic compounds has been extensively investigated in recent years,bioconversion has shown promise for its mild conditions due to the harsh chemical reaction conditions.This review discusses pro-tein engineering potential for improving glucose isomerase production and recent advancements in bioconversion of 5-hydroxymethylfurfural into value-added furanic derivatives.It suggests bi-ological strategies for the industrial transformation of 5-hydroxymethylfurfural.
