Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeuti...Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeutic effects.Low molecular weight heparin(LMWH)is a highly active low molecular weight fragment obtained via enzymatic reaction or the chemical degradation of heparin.LMWH has been applied globally in the prevention and treatment of venous thromboembolism in thrombosis patients.Simultaneously,as a potential prebiotic,because of its low molecular weight,LMWH can be well degraded by the gut microbiota to maintain intestinal balance.Enzymatic heparin degradation has recently emerged as a viable disposal method for LMWH preparation;however,only very few benchmark enzymes have been thoroughly described and subjected to protein engineering to improve their properties over the past few years.The commercialization of enzymes will require the development of robustly engineered enzymes that meet the demands of industrial processes.Herein,we report a rational protein engineering strategy that includes molecular dynamic simulations of flexible amino acid mutations and disulfide bond screening.Several Bacteroides thetaiotaomicron heparanase I(Bt-HepI)mutants were obtained and screened for high thermal stability.We obtained the Bt-HepI^(D204C/K208C/H189W/Q198R)variant,which features a stabilized protein surface structure,with a 1.3-fold increase in catalytic constant/michaelis-menten constant(k_(cat)/K_(m)),a 2.44-fold increase in thermal stability at 50℃,and a 1.8-fold decrease in the average molecular weight of LMWH produced at 40℃compared with that seen with Bt-HepI^(WT).Our study establishes a strategy to engineer thermostable HepI to underpin its industrial applications.展开更多
The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes.Microbial keratinases can convert keratin wastes...The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes.Microbial keratinases can convert keratin wastes into valuable compounds for mankind.Since the market for keratinases cannot be satisfied by production from wild hosts,it is obligatory to develop hosts with high keratinase yields.The intention of this review is to evaluate microbial keratinase advancement through protein engineering,breeding techniques,and fermentation optimization.The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods.Moreover,modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield.Intending to accelerate the production of modified keratinase,this review attempts to highlight the optimization of expression elements,such as promoter engineering,UTR,signal peptide,and codon optimization.Moreover,the approaches of host engineering including strengthening precursor supply,membrane surface engineering,and optimization of secretion pathways were also explained here.Furthermore,it is also essential to optimize the medium composition and fermentation condition for high keratinase yield.This review also addressed the present advancements,difficulties,and tendencies in the field of microbial keratinase production,along with its potential.展开更多
Longifolene(C15H24)is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications.Traditional extraction methods from plants suffer from low titer and lengthy production...Longifolene(C15H24)is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications.Traditional extraction methods from plants suffer from low titer and lengthy production cycles,while chemical synthesis is hampered by the compound’s complex structure,leading to high costs and insufficient market supply.This study aimed to develop a microbial cell factory for enhanced longifolene production.The strategy involved integrating longifolene synthase from Pinus sylvestris(PsTPS)into Yarrowia lipolytica and employing multiple metabolic engineering approaches.Initially,key genes in the mevalonate(MVA)pathway were overexpressed to enhance longifolene precursor availability for longifolene biosynthesis.Subsequently,protein engineering techniques were applied to optimize PsTPS(tPsTPS)for improved catalytic efficiency.Furthermore,co-expression of molecular chaperones was implemented to enhance the synthesis and secretion of PsTPS.The introduction of the isopentenol utilization pathway(IUP)further augmented the supply of C5 substrate.By optimizing the culture conditions,including a reduction in culture temperature,the efflux of longifolene was increased,and the dissolved oxygen levels were enhanced to promote the growth of the strain.These collective efforts resulted culminated in the engineered strain Z03 achieving a noteworthy production level of 34.67 mg/L of longifolene in shake flasks.This study not only demonstrates the feasibility of enhancing sesquiterpene production in Y.lipolytica but also highlights the potential of microbial platforms in meeting industrial demands for complex natural products.展开更多
Recently,a novel protein language model(PLM)was published by Liang Hong group in Science Advances1,introducing PRIME(PRotein language model for Intelligent Masked pretraining and Environment prediction,Fig.1).PRIME is...Recently,a novel protein language model(PLM)was published by Liang Hong group in Science Advances1,introducing PRIME(PRotein language model for Intelligent Masked pretraining and Environment prediction,Fig.1).PRIME is a deep learning model designed to predict and improve protein stability and activity without relying on experimental mutagenesis data.This innovative approach leverages a vast dataset of 96 million protein sequences annotated with their host bacterial optimal growth temperatures(OGTs)to develop a model that effectively guides protein engineering across various applications.展开更多
Natural products have garnered significant attention due to their exceptional industrial and medicinal value.Glycosylation,a crucial structural modification in these products,is typically mediated by uridine diphospha...Natural products have garnered significant attention due to their exceptional industrial and medicinal value.Glycosylation,a crucial structural modification in these products,is typically mediated by uridine diphosphate-dependent glycosyltransferases(UGTs).These enzymes not only enhance the physicochemical properties of natural products but also markedly increase their biological activity.This review provides an in-depth exploration of the characteristic features of UGTs and their diverse applications in the glycosylation modification of natural products,encompassing terpenoids,flavonoids,alkaloids,phenolic acids,and amide compounds.Special emphasis has been placed on the latest strategies in UGTs’protein engineering and high-throughput screening methods,which provides insights into the study of UGTs’protein engineering and facilitate their application in the fields of food and medicine.展开更多
Advances in deep learning have significantly aided protein engineering in addressing challenges in industrial production,healthcare,and environmental sustainability.This review frames frequently researched problems in...Advances in deep learning have significantly aided protein engineering in addressing challenges in industrial production,healthcare,and environmental sustainability.This review frames frequently researched problems in protein understanding and engineering from the perspective of deep learning.It provides a thorough discussion of representation methods for protein sequences and structures,along with general encoding pipelines that support both pre‐training and supervised learning tasks.We summarize state‐of‐the‐art protein language models,geometric deep learning techniques,and the combination of distinct approaches to learning from multi‐modal biological data.Additionally,we outline common downstream tasks and relevant benchmark datasets for training and evaluating deep learning models,focusing on satisfying the particular needs of protein engineering applications,such as identifying mutation sites and predicting properties for candidates'virtual screening.This review offers biologists the latest tools for assisting their engineering projects while providing a clear and comprehensive guide for computer scientists to develop more powerful solutions by standardizing problem formulation and consolidating data resources.Future research can foresee a deeper integration of the communities of biology and computer science,unleashing the full potential of deep learning in protein engineering and driving new scientific breakthroughs.展开更多
Comprehensive Summary The implementation of divergent protein engineering on the natural transaminase Vf-ω-TA led to the development of two effective mutants(M2 and M8),enabling the enzymatic synthesis of chiral amin...Comprehensive Summary The implementation of divergent protein engineering on the natural transaminase Vf-ω-TA led to the development of two effective mutants(M2 and M8),enabling the enzymatic synthesis of chiral amine precursors of Rivastigmine and Apremilast,respectively.The evolution of the enzymes was guided by crystal structures and a focused mutagenesis strategy,allowing them to effectively address the challenging ketone substrates with significant steric hindrance.Under the optimized reaction parameters,transamination proceeded smoothly in good conversions and with perfect stereochemical control(>99%ee).These processes utilize inexpensiveα-methylbenzylamine as an amine donor and avoid the continuous acetone removal and costly LDH/GDH/NADH systems.展开更多
Controlling protein topology has been a long standing challenge to go beyond their linear configuration defined by the translation mechanism of cellular machinery. In this mini-review, we focus on the topological dive...Controlling protein topology has been a long standing challenge to go beyond their linear configuration defined by the translation mechanism of cellular machinery. In this mini-review, we focus on the topological diversity in proteins and review the major categories of protein topologies known to date, including branched/star proteins, circular proteins, lasso proteins, knotted proteins, and protein catenanes. The discovery of these topologically complex natural proteins and their synthetic pathways, the rational design and recombinant synthesis of artificial topological proteins and their biophysical studies, are summarized and discussed with regard to their general features and broad implications. The complexity of protein topology is recognized and the routes to diverse protein topologies are illustrated. We believe that topology engineering is an important way to modify protein properties without altemating their native sequences and shall bring in valuable dynamic features central to the creation of artificial protein machinery.展开更多
Two natural nicotinamide-based coenzymes(NAD and NADP)are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism,respectively.Most NAD(P)-dependent oxidoreductases prefer one coenz...Two natural nicotinamide-based coenzymes(NAD and NADP)are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism,respectively.Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles.This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways.Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering,which is closely related to product-oriented synthetic biology projects.This review focuses on the methodology of nicotinamide-based coenzyme engineering,with its application in improving product yields and decreasing production costs.Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes.Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review.Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering,especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.展开更多
Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characteri...Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their functions.We previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these enzymes.With this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active site.Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells,expressing them in heterologous hosts usually results in problems of expression or activity.Here,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active site.After screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of CYP87D20.In addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis.This study shows the importance of considering the interplay between surface and active site residues for P450 engineering.Our integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.展开更多
Triterpenoids are a class of natural products widely used in fields related to medicine and health due to their biological activities such as hepatoprotection,anti-inflammation,anti-viral,and anti-tumor.With the advan...Triterpenoids are a class of natural products widely used in fields related to medicine and health due to their biological activities such as hepatoprotection,anti-inflammation,anti-viral,and anti-tumor.With the advancement in biotechnology,microorganisms have been used as cell factories to produce diverse natural products.Despite the significant progress that has been made in the construction of microbial cell factories for the heterogeneous biosynthesis of triterpenoids,the industrial production of triterpenoids employing microorganisms has been stymied due to the shortage of efficient enzymes as well as the low expression and low catalytic activity of heterologous proteins in microbes.Protein engineering has been demonstrated as an effective way for improving the specificity,catalytic activity,and stability of the enzyme,which can be employed to overcome these challenges.This review summarizes the current progress in the studies of Oxidosqualene cyclases(OSCs),cytochrome P450s(P450s),and UDP-glycosyltransferases(UGTs),the key enzymes in the triterpenoids synthetic pathway.The main obstacles restricting the efficient catalysis of these key enzymes are analyzed,the applications of protein engineering for the three key enzymes in the microbial synthesis of triterpenoids are systematically reviewed,and the challenges and prospects of protein engineering are also discussed.展开更多
In this dispensation of the fourth industrial revolution,protein engineering has become a popular approach for increasing enzymatic activity,stability,and titer in the biosynthesis of natural products.This is attribut...In this dispensation of the fourth industrial revolution,protein engineering has become a popular approach for increasing enzymatic activity,stability,and titer in the biosynthesis of natural products.This is attributed to its numerous advantages(over direct isolation from plants or via chemical synthesis),including decreasing or eliminating reaction byproducts,high precision,moderate handling of intricate and chemically unstable chemicals,overall reusability,and cost efficiency.Recently,protein engineering tools have advanced to redesign and enhance natural product biosynthesis.These methods include direct evolution,substrate engineering,medium engineering,enzyme engineering and immobilization,structure-assisted protein engineering,and advanced computational.Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article.Also,the advantages,limitations,and applications in industrial and medical biotechnology were discussed.Last,future research directions and potential were also highlighted.展开更多
Functional lipids,primarily derived through the modification of natural lipids by various processes,are widely acknowledged for their potential to impart health benefits.In contrast to chemical methods for lipid modif...Functional lipids,primarily derived through the modification of natural lipids by various processes,are widely acknowledged for their potential to impart health benefits.In contrast to chemical methods for lipid modification,enzymatic catalysis offers distinct advantages,including high selectivity,mild operating conditions,and reduced byproduct formation.Nevertheless,enzymes face challenges in industrial applications,such as low activity,stability,and undesired selectivity.To address these challenges,protein engineering techniques have been implemented to enhance enzyme performance in functional lipid synthesis.This article aims to review recent advances in protein engineering,encompassing approaches from directed evolution to rational design,with the goal of improving the properties of lipid-modifying enzymes.Furthermore,the article explores the future prospects and challenges associated with enzyme-catalyzed functional lipid synthesis.展开更多
Protein biologics are powerful therapeutic agents with diverse inhibitory and enzymatic functions.However,their clinical use has been limited to extracellular applications due to their inability to cross plasma membra...Protein biologics are powerful therapeutic agents with diverse inhibitory and enzymatic functions.However,their clinical use has been limited to extracellular applications due to their inability to cross plasma membranes.Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases.In this review,we highlight progress made toward achieving cytosolic delivery of recombinant proteins.We start by first considering intracellular protein delivery as a drug modality compared to existing Food and Drug Administration-approved drug modalities.Then,we summarize strategies that have been reported to achieve protein internalization.These techniques can be broadly classified into 3 categories:physical methods,direct protein engineering,and nanocarrier-mediated delivery.Finally,we highlight existing challenges for cytosolic protein delivery and offer an outlook for future advances.展开更多
N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imi...N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imine reductase from Streptomyces albidoflavus(SaIRED)for the reductive amination of biobased furans.A simple,fast and interference-resistant high-throughput screening(HTS)method was developed,based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine.The reductive amination activity of IREDs can be directly indicated by a colorimetric assay.With the reductive amination of furfural with allylamine as the model reaction,SaIRED with the activity of 4.8 U mg^(-1) was subjected to three rounds of protein engineering and screening by this HTS method,affording a high-activity tri-variant I127V/D241A/A242T(named M3,20.2 U mg^(-1)).The variant M3 showed broad substrate scope,and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines,giving the target FAs in yields up to>99%.In addition,other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method,with isolated yields up to 87%and turnover numbers up to 9700 for enzyme.Gram-scale synthesis of N-allyl-FA,a valuable building block and potential polymer monomer,was implemented at 0.25 mol L^(-1) substrate loading by a whole-cell catalyst incorporating variant M3,with 4.7 g L^(-1) h^(-1) space-time yield and 91%isolated yield.展开更多
Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This un...Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This unique character underscores their essentiality and potential as promising tools in synthetic biology.Recent advancements in enzyme engineering have significantly enhanced the catalytic performance of both natural and artificial peroxygenases.Extensive engineering efforts have been directed towards unspecific peroxygenases and fatty acid peroxygenases,aiming to expand their substrate specificities,and enhance reaction selectivities,as well as increase enzyme stability.Furthermore,innovative strategies such as dual-functional small molecule-assisted systems and H_(2)O_(2) tunnel engineering have been harnessed to transform P450 monooxygenases into highly efficient peroxygenases,capable of catalyzing reactions with a variety of unnatural substrates.This review consolidates the latest progress in the engineered and artificial heme peroxygenases,emphasizing their catalytic performances as potent biocatalysts for sustainable organic synthesis.展开更多
Deoxynivalenol(DON),the most common mycotoxin in the world,poses serious health hazards to both humans and animals and causes significant economic losses to the food and feed industry.An intriguing method for DON deto...Deoxynivalenol(DON),the most common mycotoxin in the world,poses serious health hazards to both humans and animals and causes significant economic losses to the food and feed industry.An intriguing method for DON detoxification involves enzymatic cascade catalysis by pyrroloquinoline quinone-dependent alcohol dehydrogenase(DADH)and aldo-keto reductase(AKR13B3).However,the poor catalytic activity of AKR13B3 has limited its usefulness.Here,structure-guided steric hindrance engineering,computer-assisted protein engineering,and combinatorial mutagenesis were carried out to increase the catalytic efficiency of AKR13B3.The best mutant in this project was M28S/S65V,which outperformed wild-type AKR13B3 in terms of catalytic efficiency(kcat/Km)and specific activity by factors of 44.07 and 41.65,respectively.Kinetic parameter determination revealed that the enhanced catalytic efficiency of M28S/S65V toward 3-keto-DON was attributed to the decreased Km and increased kcat values.Moreover,the engineered enzyme was applied to degrade DON in contaminated corn steep liquor,for a 90.62%removal rate.Structure-based computational analysis provided insights into the enhanced catalytic efficiency of M28S/S65V,which was attributed to the enlarged substrate-binding pocket and reshaped pocket with a favorable hydrophilic attack distance.The results provide a reference for improving the catalytic activity of an enzyme toward non-native bulky substrates and pave the way for the development of enzymes as detoxification agents to mitigate DON in the food and feed industry.展开更多
Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromo...Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromolecular architectures provides a solution to this challenge. The past few years have witnessed the emergence of a new category of peptide-protein chemistry which can covalently stitch together protein]peptide molecules with high specificity under mild physiological conditions. It has thus inspired the concept of genetically encoded click chemistry （GECC）. As a prototype of GECC, SpyTag/ SpyCatcher chemistry has enabled the precise synthesis ofmacromolecules both in vitro and in vivo, exerting precise control over the fundamental properties of these macromolecules including length, sequence, stereochemistry and topology and leading to the creation of diverse biomaterials for a variety of applications. We thus anticipate a potential toolbox of GECC comprising multiple mutually orthogonal, covalent-bond forming peptide-protein reactive pairs with diverse features, which shall bridge synthetic biology and materials science and open up enormous opportunities for biomaterialsin the future.展开更多
Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a t...Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to ＂read＂ genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongeneticaUy by using drug.like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore; the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.展开更多
cAMP is an important second messenger that is capable of controlling a wide array of cellular processes, including glycogen, sugar and lipid metabolism. Here we report the design and construction of a novel geneticall...cAMP is an important second messenger that is capable of controlling a wide array of cellular processes, including glycogen, sugar and lipid metabolism. Here we report the design and construction of a novel genetically encoded fluorescent sensor for cAMP. The sensor was realized by fusing E. coli CAP protein with cpYFP, and displayed a one-fold fluorescence change towards c AMP binding. Further characterization assays demonstrated that the sensor had high affinity for cAMP and fast response kinetics.The development of our sensor could be a useful supplement to existing methods for cAMP detection.展开更多
基金supported by the Natural Science Foundation of Jiangsu Province(BE2021623,BK20220155)Natural Science Foundation of Jiangsu Province(BE2021623)+4 种基金National Natural Science Foundation of China(32001665,U1903205,32021005)the National Key Research and Development Program of China(2017YF0400303)the Key Scientific and Technological Research Projects in the Key Areas of the Xinjiang Production and Construction Corps(2018AB010)the Key Research and Development 303 Program of Ningxia(2020BFG02012)Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province。
文摘Heparin,a glycosaminoglycan,is a stable source of carbon that supports the growth of microorganisms in the human intestine.It is also a commonly used anticoagulant drug in clinical practice,with significant therapeutic effects.Low molecular weight heparin(LMWH)is a highly active low molecular weight fragment obtained via enzymatic reaction or the chemical degradation of heparin.LMWH has been applied globally in the prevention and treatment of venous thromboembolism in thrombosis patients.Simultaneously,as a potential prebiotic,because of its low molecular weight,LMWH can be well degraded by the gut microbiota to maintain intestinal balance.Enzymatic heparin degradation has recently emerged as a viable disposal method for LMWH preparation;however,only very few benchmark enzymes have been thoroughly described and subjected to protein engineering to improve their properties over the past few years.The commercialization of enzymes will require the development of robustly engineered enzymes that meet the demands of industrial processes.Herein,we report a rational protein engineering strategy that includes molecular dynamic simulations of flexible amino acid mutations and disulfide bond screening.Several Bacteroides thetaiotaomicron heparanase I(Bt-HepI)mutants were obtained and screened for high thermal stability.We obtained the Bt-HepI^(D204C/K208C/H189W/Q198R)variant,which features a stabilized protein surface structure,with a 1.3-fold increase in catalytic constant/michaelis-menten constant(k_(cat)/K_(m)),a 2.44-fold increase in thermal stability at 50℃,and a 1.8-fold decrease in the average molecular weight of LMWH produced at 40℃compared with that seen with Bt-HepI^(WT).Our study establishes a strategy to engineer thermostable HepI to underpin its industrial applications.
基金supported by the National Key Research and Development Program of China(2021YFC2100202)Science and Technology Project of Hubei Tobacco Company(027Y2021-023).
文摘The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes.Microbial keratinases can convert keratin wastes into valuable compounds for mankind.Since the market for keratinases cannot be satisfied by production from wild hosts,it is obligatory to develop hosts with high keratinase yields.The intention of this review is to evaluate microbial keratinase advancement through protein engineering,breeding techniques,and fermentation optimization.The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods.Moreover,modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield.Intending to accelerate the production of modified keratinase,this review attempts to highlight the optimization of expression elements,such as promoter engineering,UTR,signal peptide,and codon optimization.Moreover,the approaches of host engineering including strengthening precursor supply,membrane surface engineering,and optimization of secretion pathways were also explained here.Furthermore,it is also essential to optimize the medium composition and fermentation condition for high keratinase yield.This review also addressed the present advancements,difficulties,and tendencies in the field of microbial keratinase production,along with its potential.
基金supported by the National Natural Science Foundation of China(No.42206137,32270118)the Science and Technology Planning Project of Guangzhou(2024A04J4129)the Natural Science Foundation of Guangdong Province(No.2019B1515120062).
文摘Longifolene(C15H24)is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications.Traditional extraction methods from plants suffer from low titer and lengthy production cycles,while chemical synthesis is hampered by the compound’s complex structure,leading to high costs and insufficient market supply.This study aimed to develop a microbial cell factory for enhanced longifolene production.The strategy involved integrating longifolene synthase from Pinus sylvestris(PsTPS)into Yarrowia lipolytica and employing multiple metabolic engineering approaches.Initially,key genes in the mevalonate(MVA)pathway were overexpressed to enhance longifolene precursor availability for longifolene biosynthesis.Subsequently,protein engineering techniques were applied to optimize PsTPS(tPsTPS)for improved catalytic efficiency.Furthermore,co-expression of molecular chaperones was implemented to enhance the synthesis and secretion of PsTPS.The introduction of the isopentenol utilization pathway(IUP)further augmented the supply of C5 substrate.By optimizing the culture conditions,including a reduction in culture temperature,the efflux of longifolene was increased,and the dissolved oxygen levels were enhanced to promote the growth of the strain.These collective efforts resulted culminated in the engineered strain Z03 achieving a noteworthy production level of 34.67 mg/L of longifolene in shake flasks.This study not only demonstrates the feasibility of enhancing sesquiterpene production in Y.lipolytica but also highlights the potential of microbial platforms in meeting industrial demands for complex natural products.
文摘Recently,a novel protein language model(PLM)was published by Liang Hong group in Science Advances1,introducing PRIME(PRotein language model for Intelligent Masked pretraining and Environment prediction,Fig.1).PRIME is a deep learning model designed to predict and improve protein stability and activity without relying on experimental mutagenesis data.This innovative approach leverages a vast dataset of 96 million protein sequences annotated with their host bacterial optimal growth temperatures(OGTs)to develop a model that effectively guides protein engineering across various applications.
基金supported by the National Natural Science Foundation of China(No.32172192)the Zhejiang Provincial Natural Science Foundation of China(No.ZCLQ24C2001).
文摘Natural products have garnered significant attention due to their exceptional industrial and medicinal value.Glycosylation,a crucial structural modification in these products,is typically mediated by uridine diphosphate-dependent glycosyltransferases(UGTs).These enzymes not only enhance the physicochemical properties of natural products but also markedly increase their biological activity.This review provides an in-depth exploration of the characteristic features of UGTs and their diverse applications in the glycosylation modification of natural products,encompassing terpenoids,flavonoids,alkaloids,phenolic acids,and amide compounds.Special emphasis has been placed on the latest strategies in UGTs’protein engineering and high-throughput screening methods,which provides insights into the study of UGTs’protein engineering and facilitate their application in the fields of food and medicine.
基金supported by the National Natural Science Foundation of China(11974239 and 62302291)Innovation Program of Shanghai Municipal Education Commission(2019‐01‐07‐00‐02‐E00076)Shanghai Jiao Tong University Scientific and Technological Innovation Funds(21×010200843)。
文摘Advances in deep learning have significantly aided protein engineering in addressing challenges in industrial production,healthcare,and environmental sustainability.This review frames frequently researched problems in protein understanding and engineering from the perspective of deep learning.It provides a thorough discussion of representation methods for protein sequences and structures,along with general encoding pipelines that support both pre‐training and supervised learning tasks.We summarize state‐of‐the‐art protein language models,geometric deep learning techniques,and the combination of distinct approaches to learning from multi‐modal biological data.Additionally,we outline common downstream tasks and relevant benchmark datasets for training and evaluating deep learning models,focusing on satisfying the particular needs of protein engineering applications,such as identifying mutation sites and predicting properties for candidates'virtual screening.This review offers biologists the latest tools for assisting their engineering projects while providing a clear and comprehensive guide for computer scientists to develop more powerful solutions by standardizing problem formulation and consolidating data resources.Future research can foresee a deeper integration of the communities of biology and computer science,unleashing the full potential of deep learning in protein engineering and driving new scientific breakthroughs.
基金the National Key R&D Program of China(No.2021YFF1200203 to G.W.and 2018YFA0903500 to F.Z.)Hubei Provincial Key R&D program(2021BAA168 to Y.W.)+2 种基金Shen-Zhen Science and Technology Program(JCYJ20220530160805011 to F.Z.)the interdisciplinary research program of Huazhong University of Science and Technology(HUST)(2023JCYJ001 to F.Z.)the China Postdoctoral Science Foundation(2023M741259 to X.Y.)for financial supports.
文摘Comprehensive Summary The implementation of divergent protein engineering on the natural transaminase Vf-ω-TA led to the development of two effective mutants(M2 and M8),enabling the enzymatic synthesis of chiral amine precursors of Rivastigmine and Apremilast,respectively.The evolution of the enzymes was guided by crystal structures and a focused mutagenesis strategy,allowing them to effectively address the challenging ketone substrates with significant steric hindrance.Under the optimized reaction parameters,transamination proceeded smoothly in good conversions and with perfect stereochemical control(>99%ee).These processes utilize inexpensiveα-methylbenzylamine as an amine donor and avoid the continuous acetone removal and costly LDH/GDH/NADH systems.
基金supported by the National High Technology Research and Development Program of China (2015AA020941)the National Natural Science Foundation of China (21474003, 91427304)"1000 Plan (Youth)"
文摘Controlling protein topology has been a long standing challenge to go beyond their linear configuration defined by the translation mechanism of cellular machinery. In this mini-review, we focus on the topological diversity in proteins and review the major categories of protein topologies known to date, including branched/star proteins, circular proteins, lasso proteins, knotted proteins, and protein catenanes. The discovery of these topologically complex natural proteins and their synthetic pathways, the rational design and recombinant synthesis of artificial topological proteins and their biophysical studies, are summarized and discussed with regard to their general features and broad implications. The complexity of protein topology is recognized and the routes to diverse protein topologies are illustrated. We believe that topology engineering is an important way to modify protein properties without altemating their native sequences and shall bring in valuable dynamic features central to the creation of artificial protein machinery.
基金This study was mainly supported by the Key Research Program of the Chinese Academy of Sciences(Grant No.ZDRW-ZS-2016-3)1000-youth program of China to CY and the National Natural Science Foundation of China(Grant No.31600636)Funds were partially provided by the DOE EERE award(DE-EE0006968)to YPZ.
文摘Two natural nicotinamide-based coenzymes(NAD and NADP)are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism,respectively.Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles.This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways.Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering,which is closely related to product-oriented synthetic biology projects.This review focuses on the methodology of nicotinamide-based coenzyme engineering,with its application in improving product yields and decreasing production costs.Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes.Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review.Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering,especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.
基金the National Key Research and Development Program of China(2018YFA0901800)Yunnan Science Fund(202005AE160015 and 2019FJ004)This work was also supported from Shenzhen Municipal Governments.
文摘Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their functions.We previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these enzymes.With this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active site.Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells,expressing them in heterologous hosts usually results in problems of expression or activity.Here,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active site.After screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of CYP87D20.In addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis.This study shows the importance of considering the interplay between surface and active site residues for P450 engineering.Our integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.
基金support from the National Key Research and Development Program of China (2019YFA0905700,2018YFA0901800)the National Natural Science Foundation of China (22078020)Young Elite Scientists Sponsorship Program by CAST (2019QNRC001).
文摘Triterpenoids are a class of natural products widely used in fields related to medicine and health due to their biological activities such as hepatoprotection,anti-inflammation,anti-viral,and anti-tumor.With the advancement in biotechnology,microorganisms have been used as cell factories to produce diverse natural products.Despite the significant progress that has been made in the construction of microbial cell factories for the heterogeneous biosynthesis of triterpenoids,the industrial production of triterpenoids employing microorganisms has been stymied due to the shortage of efficient enzymes as well as the low expression and low catalytic activity of heterologous proteins in microbes.Protein engineering has been demonstrated as an effective way for improving the specificity,catalytic activity,and stability of the enzyme,which can be employed to overcome these challenges.This review summarizes the current progress in the studies of Oxidosqualene cyclases(OSCs),cytochrome P450s(P450s),and UDP-glycosyltransferases(UGTs),the key enzymes in the triterpenoids synthetic pathway.The main obstacles restricting the efficient catalysis of these key enzymes are analyzed,the applications of protein engineering for the three key enzymes in the microbial synthesis of triterpenoids are systematically reviewed,and the challenges and prospects of protein engineering are also discussed.
基金funded by the University of Witwatersrand postdoctoral research fellowship obtained by O.Ssupported by the South African Research Chairs Initiative(SARChI)of the Department of Science and Technologythe National Research Foundation(grant 64788 to I.A.).
文摘In this dispensation of the fourth industrial revolution,protein engineering has become a popular approach for increasing enzymatic activity,stability,and titer in the biosynthesis of natural products.This is attributed to its numerous advantages(over direct isolation from plants or via chemical synthesis),including decreasing or eliminating reaction byproducts,high precision,moderate handling of intricate and chemically unstable chemicals,overall reusability,and cost efficiency.Recently,protein engineering tools have advanced to redesign and enhance natural product biosynthesis.These methods include direct evolution,substrate engineering,medium engineering,enzyme engineering and immobilization,structure-assisted protein engineering,and advanced computational.Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article.Also,the advantages,limitations,and applications in industrial and medical biotechnology were discussed.Last,future research directions and potential were also highlighted.
基金supported by Natural Science Foundation of Sichuan Province(2023NSFSC0132)Fundamental Research Funds for Central Universities of the Sichuan University(YJ202308).
文摘Functional lipids,primarily derived through the modification of natural lipids by various processes,are widely acknowledged for their potential to impart health benefits.In contrast to chemical methods for lipid modification,enzymatic catalysis offers distinct advantages,including high selectivity,mild operating conditions,and reduced byproduct formation.Nevertheless,enzymes face challenges in industrial applications,such as low activity,stability,and undesired selectivity.To address these challenges,protein engineering techniques have been implemented to enhance enzyme performance in functional lipid synthesis.This article aims to review recent advances in protein engineering,encompassing approaches from directed evolution to rational design,with the goal of improving the properties of lipid-modifying enzymes.Furthermore,the article explores the future prospects and challenges associated with enzyme-catalyzed functional lipid synthesis.
文摘Protein biologics are powerful therapeutic agents with diverse inhibitory and enzymatic functions.However,their clinical use has been limited to extracellular applications due to their inability to cross plasma membranes.Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases.In this review,we highlight progress made toward achieving cytosolic delivery of recombinant proteins.We start by first considering intracellular protein delivery as a drug modality compared to existing Food and Drug Administration-approved drug modalities.Then,we summarize strategies that have been reported to achieve protein internalization.These techniques can be broadly classified into 3 categories:physical methods,direct protein engineering,and nanocarrier-mediated delivery.Finally,we highlight existing challenges for cytosolic protein delivery and offer an outlook for future advances.
文摘N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imine reductase from Streptomyces albidoflavus(SaIRED)for the reductive amination of biobased furans.A simple,fast and interference-resistant high-throughput screening(HTS)method was developed,based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine.The reductive amination activity of IREDs can be directly indicated by a colorimetric assay.With the reductive amination of furfural with allylamine as the model reaction,SaIRED with the activity of 4.8 U mg^(-1) was subjected to three rounds of protein engineering and screening by this HTS method,affording a high-activity tri-variant I127V/D241A/A242T(named M3,20.2 U mg^(-1)).The variant M3 showed broad substrate scope,and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines,giving the target FAs in yields up to>99%.In addition,other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method,with isolated yields up to 87%and turnover numbers up to 9700 for enzyme.Gram-scale synthesis of N-allyl-FA,a valuable building block and potential polymer monomer,was implemented at 0.25 mol L^(-1) substrate loading by a whole-cell catalyst incorporating variant M3,with 4.7 g L^(-1) h^(-1) space-time yield and 91%isolated yield.
文摘Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This unique character underscores their essentiality and potential as promising tools in synthetic biology.Recent advancements in enzyme engineering have significantly enhanced the catalytic performance of both natural and artificial peroxygenases.Extensive engineering efforts have been directed towards unspecific peroxygenases and fatty acid peroxygenases,aiming to expand their substrate specificities,and enhance reaction selectivities,as well as increase enzyme stability.Furthermore,innovative strategies such as dual-functional small molecule-assisted systems and H_(2)O_(2) tunnel engineering have been harnessed to transform P450 monooxygenases into highly efficient peroxygenases,capable of catalyzing reactions with a variety of unnatural substrates.This review consolidates the latest progress in the engineered and artificial heme peroxygenases,emphasizing their catalytic performances as potent biocatalysts for sustainable organic synthesis.
基金funded by the National Natural Science Foundation of China(32272267)Jiangsu Provincial Frontier Technology Research and Development Program(BF2024073).
文摘Deoxynivalenol(DON),the most common mycotoxin in the world,poses serious health hazards to both humans and animals and causes significant economic losses to the food and feed industry.An intriguing method for DON detoxification involves enzymatic cascade catalysis by pyrroloquinoline quinone-dependent alcohol dehydrogenase(DADH)and aldo-keto reductase(AKR13B3).However,the poor catalytic activity of AKR13B3 has limited its usefulness.Here,structure-guided steric hindrance engineering,computer-assisted protein engineering,and combinatorial mutagenesis were carried out to increase the catalytic efficiency of AKR13B3.The best mutant in this project was M28S/S65V,which outperformed wild-type AKR13B3 in terms of catalytic efficiency(kcat/Km)and specific activity by factors of 44.07 and 41.65,respectively.Kinetic parameter determination revealed that the enhanced catalytic efficiency of M28S/S65V toward 3-keto-DON was attributed to the decreased Km and increased kcat values.Moreover,the engineered enzyme was applied to degrade DON in contaminated corn steep liquor,for a 90.62%removal rate.Structure-based computational analysis provided insights into the enhanced catalytic efficiency of M28S/S65V,which was attributed to the enlarged substrate-binding pocket and reshaped pocket with a favorable hydrophilic attack distance.The results provide a reference for improving the catalytic activity of an enzyme toward non-native bulky substrates and pave the way for the development of enzymes as detoxification agents to mitigate DON in the food and feed industry.
基金financial supports from the Research Grants Council of Hong Kong SAR Government to F. Sun (RGC-ECS Nos. #26103915 and Ao E/M-09/12)the 863 Program (No. 2015AA020941)+2 种基金the National Natural Science Foundation of China (Nos. 21474003, 91427304)"1000 Plan (Youth)"the Department of Chemical and Biological Engineering, HKUST for the faculty start-up fund
文摘Synthesis of macromolecular systems with precise structural and functional control constitutes a fundamental challenge for materials science and engineering. Development of the ability to construct complex bio-macromolecular architectures provides a solution to this challenge. The past few years have witnessed the emergence of a new category of peptide-protein chemistry which can covalently stitch together protein]peptide molecules with high specificity under mild physiological conditions. It has thus inspired the concept of genetically encoded click chemistry （GECC）. As a prototype of GECC, SpyTag/ SpyCatcher chemistry has enabled the precise synthesis ofmacromolecules both in vitro and in vivo, exerting precise control over the fundamental properties of these macromolecules including length, sequence, stereochemistry and topology and leading to the creation of diverse biomaterials for a variety of applications. We thus anticipate a potential toolbox of GECC comprising multiple mutually orthogonal, covalent-bond forming peptide-protein reactive pairs with diverse features, which shall bridge synthetic biology and materials science and open up enormous opportunities for biomaterialsin the future.
文摘Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to ＂read＂ genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongeneticaUy by using drug.like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore; the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.
基金National Basic Research Foundation of China(Grant No.2017YFA0505202)the National Natural Science Foundation of China(Grant No.91853107)
文摘cAMP is an important second messenger that is capable of controlling a wide array of cellular processes, including glycogen, sugar and lipid metabolism. Here we report the design and construction of a novel genetically encoded fluorescent sensor for cAMP. The sensor was realized by fusing E. coli CAP protein with cpYFP, and displayed a one-fold fluorescence change towards c AMP binding. Further characterization assays demonstrated that the sensor had high affinity for cAMP and fast response kinetics.The development of our sensor could be a useful supplement to existing methods for cAMP detection.
