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
Astaxanthin is a natural red carotenoid,commonly used as an additive in the pharmaceutical industry and as a nutritional supplement owing to its notable antioxidant benefits.However,a complex biosynthetic pathway pose...Astaxanthin is a natural red carotenoid,commonly used as an additive in the pharmaceutical industry and as a nutritional supplement owing to its notable antioxidant benefits.However,a complex biosynthetic pathway poses a challenge to de novo biosynthesis of astaxanthin.Here,Yarrowia lipolytica was engineered through multiple strategies for high level production of astaxanthin using a cheap mineral medium.For the production ofβ-carotene,a platform strain was constructed in which 411.7 mg/L ofβ-carotene was produced at a shake-flask level.Integration of algalβ-carotene ketolase andβ-carotene hydroxylase led to the production of 12.3 mg/L of astaxanthin.Furthermore,construction of HpBKT and HpCrtZ as a single enzyme complex along with the enhanced catalytic activity of the enzymes led to the accumulation of 41.0 mg/L of astaxanthin.Iterative gene integration into the genome and direction of the astaxanthin production pathway into sub-organelles substan-tially increased astaxanthin production(172.1 mg/L).Finally,restoration of the auxotrophic markers and me-dium optimization further improved astaxanthin production to 237.3 mg/L.The aforementioned approaches were employed in fed-batch fermentation to produce 2820 mg/L of astaxanthin(229-fold improvement regarding the starter strain),with an average productivity of 434 mg/L/d and a yield of 5.6 mg/g glucose,which is the highest reported productivity in Y.lipolytica.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
Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine...Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.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
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
Microbial catalysts offer compelling advantages for oxygen reduction reaction(ORR)in microbial fuel cell(MFC)cathodes,including reduced costs and extended operational lifespans.However,their practical application rema...Microbial catalysts offer compelling advantages for oxygen reduction reaction(ORR)in microbial fuel cell(MFC)cathodes,including reduced costs and extended operational lifespans.However,their practical application remains limited by insufficient intrinsic activity at catalytic protein sites and restricted charge accessibility,both of which constrain ORR kinetics.Here,we report the development of an efficient trifunctional bioendogenous system based on menaquinone-7(MK-7),enriched from Bacillus subtilis natto(natto digester strain(ND))through a straightforward fermentation strategy.The engineered MK-7 simultaneously performs three critical functions:(i)facilitating mediated electron transfer between bacteria and electrodes,(ii)regulating the in-situ formation of size-controlled conductive polydopamine nanostructures that enhance direct electron transfer pathways,and(iii)modulating the electronic structure of cytochrome c(Cyt c)to activate its catalytic center and optimize O_(2)adsorption capacity.Through these synergistic effects,our engineered nano-hybrid ND-FM@sPDA(FM is fermentation and sPDA is size-controlled conductive polydopamine)achieves an oxygen reduction current density of 3.83 mA·cm^(-2),representing a 1.54-fold enhancement over pristine ND(2.48 mA·cm^(-2)).MFCs constructed with the ND-FM@sPDA biocathode deliver a peak power density of 412μW·cm^(-2),surpassing previously reported microbial catalysts for similar applications.This work elucidates novel regulatory mechanisms for optimizing biocatalysts at the molecular level and provides critical insights for advancing sustainable bioelectrocatalytic technologies with enhanced performance.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 b...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
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
δ-Tocotrienol,an isomer of vitamin E with anti-inflammatory,neuroprotective and anti-coronary arteriosclerosis properties,is widely used in health care,medicine and other fields.Microbial synthesis ofδ-tocotrienol o...δ-Tocotrienol,an isomer of vitamin E with anti-inflammatory,neuroprotective and anti-coronary arteriosclerosis properties,is widely used in health care,medicine and other fields.Microbial synthesis ofδ-tocotrienol offers significant advantages over plant extraction and chemical synthesis methods,including increased efficiency,cost-effectiveness and environmental sustainability.However,limited precursor availability and low catalytic efficiency of key enzymes remain major bottlenecks in the biosynthesis ofδ-tocotrienol.In this study,we assembled the completeδ-tocotrienol biosynthetic pathway in Yarrowia lipolytica and enhanced the precursor supply,resulting in a titre of 102.8 mg/L.The catalytic efficiency of the rate-limiting steps in the pathway was then enhanced through various strategies,including fusion expression of key enzymes homogentisate phytyl-transferaseand and tocopherol cyclase,semi-rational design of SyHPT and multi-copy integration of pathway genes.The final aδ-tocotrienol titre in a 5 L bioreactor was 466.8 mg/L following fed-batchfermentation.This study represents the first successful de novo biosynthesis ofδ-tocotrienol in Y.lipolytica,providing valuable in-sights into the synthesis of vitamin E-related compounds.展开更多
Parthenolide is confirmed to be an important component of the anticancer drug-ACT001.However,parthe-nolide biosynthesis in Saccharomyces cerevisiae(S.cerevisiae)was greatly hindered by the low conversion rate of its p...Parthenolide is confirmed to be an important component of the anticancer drug-ACT001.However,parthe-nolide biosynthesis in Saccharomyces cerevisiae(S.cerevisiae)was greatly hindered by the low conversion rate of its precursor,costunolide.In this study,the Position Specific Scoring Matrix(PSSM)was used to analyze the sequence evolutionary information of parthenolide synthase from Tanacetum parthenium(TpPTS),and a series of mutants were designed and validated.Notably,when the mutant of TpPTS-Y22G was introduced in S.cerevisiae,the parthenolide titer increased by 110%compared to that of the TpPTS wild-type.Considering TpPTS as an endoplasmic reticulum-localized cytochrome P450 and the importance of heme supply,endoplasmic-associated molecular chaperone HRD1(hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1)and heme biosynthesis gene HEM2(aminolevulinate dehydratase)were overexpressed in S.cerevisiae to improve TpPTS expression and catalytic activity.As a result,a titer of 27.08 mg/L parthenolide was achieved in a shake flask,which was further increased by 209%.Additionally,the conversion rate of costunolide to parthenolide increased from 20.4%to 51.8%compared to the initial strain yYTQ001.Eventually,a parthenolide titer of 99.71 mg/L was achieved in a 5-L bioreactor.Our research provides effective strategies and valuable references for engi-neering rate-limiting cytochrome P450 enzymes to improve sesquiterpenes production in S.cerevisiae.展开更多
The rapid production of nonbiodegradable plastics has led to a sig-nificant increase in plastic waste.A shift toward biodegradable and compostable plastics has become inevitable.
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
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
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supported by the National Key Research and Devel-opment Program of China(2022YFC2106100)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005).
文摘Astaxanthin is a natural red carotenoid,commonly used as an additive in the pharmaceutical industry and as a nutritional supplement owing to its notable antioxidant benefits.However,a complex biosynthetic pathway poses a challenge to de novo biosynthesis of astaxanthin.Here,Yarrowia lipolytica was engineered through multiple strategies for high level production of astaxanthin using a cheap mineral medium.For the production ofβ-carotene,a platform strain was constructed in which 411.7 mg/L ofβ-carotene was produced at a shake-flask level.Integration of algalβ-carotene ketolase andβ-carotene hydroxylase led to the production of 12.3 mg/L of astaxanthin.Furthermore,construction of HpBKT and HpCrtZ as a single enzyme complex along with the enhanced catalytic activity of the enzymes led to the accumulation of 41.0 mg/L of astaxanthin.Iterative gene integration into the genome and direction of the astaxanthin production pathway into sub-organelles substan-tially increased astaxanthin production(172.1 mg/L).Finally,restoration of the auxotrophic markers and me-dium optimization further improved astaxanthin production to 237.3 mg/L.The aforementioned approaches were employed in fed-batch fermentation to produce 2820 mg/L of astaxanthin(229-fold improvement regarding the starter strain),with an average productivity of 434 mg/L/d and a yield of 5.6 mg/g glucose,which is the highest reported productivity in Y.lipolytica.
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.
基金supported by the National Key Research and Development Program of China(2020YFA0908300)the Natural Science Foundation of China(22138006,22278240).
文摘Terpenoids,one of the most diverse and structurally varied natural products in nature,are widely distributed in plants,microbes,and other organisms.Their structural diversity confers significant importance in medicine,food,flavorings,and energy.However,traditional methods of plant extraction and chemical synthesis have limitations in industrial applications.Consequently,microbial cell factories have emerged as an important platform for terpenoid production.Terpene synthases(TPSs)are crucial in determining the structural and functional diversity of terpenoids.This review discussed the origin and classificationof TPSs,outlines commonly used TPS mining methods,and summarizes advances in TPS engineering.In addition,it also explores the influenceof machine learning on enzyme mining,the existing challenges and the future opportunities alongside cutting-edge technologies.
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.
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supported by the National Natural Science Foundation of China(Nos.52472200 and 52271176)the 111 Project(No.D17007)+1 种基金the Henan Center for Outstanding Overseas Scientists(No.GZS2022017)Henan Province Key Research and Development Project(No.231111520500).
文摘Microbial catalysts offer compelling advantages for oxygen reduction reaction(ORR)in microbial fuel cell(MFC)cathodes,including reduced costs and extended operational lifespans.However,their practical application remains limited by insufficient intrinsic activity at catalytic protein sites and restricted charge accessibility,both of which constrain ORR kinetics.Here,we report the development of an efficient trifunctional bioendogenous system based on menaquinone-7(MK-7),enriched from Bacillus subtilis natto(natto digester strain(ND))through a straightforward fermentation strategy.The engineered MK-7 simultaneously performs three critical functions:(i)facilitating mediated electron transfer between bacteria and electrodes,(ii)regulating the in-situ formation of size-controlled conductive polydopamine nanostructures that enhance direct electron transfer pathways,and(iii)modulating the electronic structure of cytochrome c(Cyt c)to activate its catalytic center and optimize O_(2)adsorption capacity.Through these synergistic effects,our engineered nano-hybrid ND-FM@sPDA(FM is fermentation and sPDA is size-controlled conductive polydopamine)achieves an oxygen reduction current density of 3.83 mA·cm^(-2),representing a 1.54-fold enhancement over pristine ND(2.48 mA·cm^(-2)).MFCs constructed with the ND-FM@sPDA biocathode deliver a peak power density of 412μW·cm^(-2),surpassing previously reported microbial catalysts for similar applications.This work elucidates novel regulatory mechanisms for optimizing biocatalysts at the molecular level and provides critical insights for advancing sustainable bioelectrocatalytic technologies with enhanced performance.
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 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supported by the National Key Research and Devel-opment Program of China(2023YFF1103700)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005).
文摘δ-Tocotrienol,an isomer of vitamin E with anti-inflammatory,neuroprotective and anti-coronary arteriosclerosis properties,is widely used in health care,medicine and other fields.Microbial synthesis ofδ-tocotrienol offers significant advantages over plant extraction and chemical synthesis methods,including increased efficiency,cost-effectiveness and environmental sustainability.However,limited precursor availability and low catalytic efficiency of key enzymes remain major bottlenecks in the biosynthesis ofδ-tocotrienol.In this study,we assembled the completeδ-tocotrienol biosynthetic pathway in Yarrowia lipolytica and enhanced the precursor supply,resulting in a titre of 102.8 mg/L.The catalytic efficiency of the rate-limiting steps in the pathway was then enhanced through various strategies,including fusion expression of key enzymes homogentisate phytyl-transferaseand and tocopherol cyclase,semi-rational design of SyHPT and multi-copy integration of pathway genes.The final aδ-tocotrienol titre in a 5 L bioreactor was 466.8 mg/L following fed-batchfermentation.This study represents the first successful de novo biosynthesis ofδ-tocotrienol in Y.lipolytica,providing valuable in-sights into the synthesis of vitamin E-related compounds.
基金funded by the National Key Research and Develop-ment Program(2024YFA0917100)the Key-Area Research and Development Program of Guangdong Province(2020B0303070002)the National Natural Science Foundation of China(22178261).
文摘Parthenolide is confirmed to be an important component of the anticancer drug-ACT001.However,parthe-nolide biosynthesis in Saccharomyces cerevisiae(S.cerevisiae)was greatly hindered by the low conversion rate of its precursor,costunolide.In this study,the Position Specific Scoring Matrix(PSSM)was used to analyze the sequence evolutionary information of parthenolide synthase from Tanacetum parthenium(TpPTS),and a series of mutants were designed and validated.Notably,when the mutant of TpPTS-Y22G was introduced in S.cerevisiae,the parthenolide titer increased by 110%compared to that of the TpPTS wild-type.Considering TpPTS as an endoplasmic reticulum-localized cytochrome P450 and the importance of heme supply,endoplasmic-associated molecular chaperone HRD1(hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1)and heme biosynthesis gene HEM2(aminolevulinate dehydratase)were overexpressed in S.cerevisiae to improve TpPTS expression and catalytic activity.As a result,a titer of 27.08 mg/L parthenolide was achieved in a shake flask,which was further increased by 209%.Additionally,the conversion rate of costunolide to parthenolide increased from 20.4%to 51.8%compared to the initial strain yYTQ001.Eventually,a parthenolide titer of 99.71 mg/L was achieved in a 5-L bioreactor.Our research provides effective strategies and valuable references for engi-neering rate-limiting cytochrome P450 enzymes to improve sesquiterpenes production in S.cerevisiae.
基金supported by the Science and Technology Development Plan Project of Jilin Province,China(Grant No.20230203159SF).
文摘The rapid production of nonbiodegradable plastics has led to a sig-nificant increase in plastic waste.A shift toward biodegradable and compostable plastics has become inevitable.
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.
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.
