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
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
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
Enhancing the stability of biomolecules is one of the hot topics in industry.In this study,we enhanced the stability of an important protein called LEPTIN.LEPTIN is a hormone secreted by fat cells playing an essential...Enhancing the stability of biomolecules is one of the hot topics in industry.In this study,we enhanced the stability of an important protein called LEPTIN.LEPTIN is a hormone secreted by fat cells playing an essential role in body weight and composition,and its deficiency can result in several disorders.The treatment of related LEPTIN dysfunctions is often available in the form of injection.To decrease the cost and the frequency of its applications can be achieved by increasing its lifetime through engineering LEPTIN.In this study,to engineer LEPTIN,we have introduced disulfide bonds.Disulfide By Design server was used to predict the suitable nominate pairs,which suggested three pairs of amino acids to be mutated to cysteine for disulfide bond formation.Additionally,to further evaluate the effect of combined mutations,we combined these three nominated pairs to produce three more mutants.In order to assess the effect of introduced mutations,molecular dynamic(MD)simulation was performed.The result suggests that Mutant-1 is more stable in comparison to wild-type and the other mutants.Moreover,docking results showed that the introduced mutation does not affect the receptor binding performance;therefore,it can be considered a suitable choice for future protein engineering.展开更多
The deep-learning protein structure prediction method AlphaFold2 has garnered enormous attention beyond the realm of structural biology,for its groundbreaking contribution to solving the"protein foiding problem&q...The deep-learning protein structure prediction method AlphaFold2 has garnered enormous attention beyond the realm of structural biology,for its groundbreaking contribution to solving the"protein foiding problem"In this perspective,we explore the connection between protein structure studies and environmental research,delving into the potential for addressing specific environmental challenges.Proteins are promising for environmental applications because of the functional diversity endowed by their structural complexity.However,structural studies on proteins with environmental significance remain scarce.Here,we present the opportunity to study proteins by advancing experimental determination and deep-learning prediction methods.Specifically,the latest progress in environmental research via cryogenic electron microscopy is highlighted.It allows us to determine the structure of protein complexes in their native state within cells at molecular resolution,revealing environmentally-associated structural dynamics.With the remarkable advancements in computational power and experimental resolution,the study of protein structure and dynamics has reached unprecedented depth and accuracy.These advancements will undoubtedly accelerate the establishment of comprehensive environmental protein structural and functional databases.Tremendous opportunities for protein engineering exist to enable innovative solutions for environmental applications,such as the degradation of persistent contaminants,and the recovery of valuable metals as well as rare earth elements.展开更多
L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the p...L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.展开更多
Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield a...Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield and high cost.Microbial production represents as a promising alternative for efficient and sustainable production of PCs.In this review,we summarize recent advances in this field,which include enzyme mining and engineering to construct artificial pathways,balance of enzyme expression to improve pathway efficiency,coculture engineering to alleviate metabolic burden and side-reactions,and the use of genetic circuits for dynamic regulation and high throughput screening.Finally,current challenges and future perspectives for efficient production of PCs are also discussed.展开更多
BACKGROUND:The incidence of hepatocellular carcinoma (HCC)in China is closely related to the population infected with hepatitis B virus(HBV).HCC cells with HBV secrete soluble HBsAg into blood but do not express it on...BACKGROUND:The incidence of hepatocellular carcinoma (HCC)in China is closely related to the population infected with hepatitis B virus(HBV).HCC cells with HBV secrete soluble HBsAg into blood but do not express it on the cell membrane This study aimed to construct and investigate a new glycosyl phosphatidylinositol(GPI)-anchored protein(GPC3+α+EGFP) as a DNA vaccine against HCC associated with HBV. METHODS:A recombinant plasmid(pcDNA3.1(+)/GPC3+ α+EGFP)was constructed and verified by restriction endo nuclease digestion and sequencing.pcDNA3.1(+)/GPC3+α+ EGFP was transfected into HepG2 cells(experimental group) using lipofectamine 2000.pEGFP-N1-transfected HepG2 cells were used as a negative control,and non-transfected HepG2 cells sreved as a blank control.HepG2 cells that steadily expressed the fusion protein GPC3+α+EGFP were screened by G418,propagated,and co-cultured with lymphocytes from healthy donors.Cell proliferation was measured by the classic sulforhodamine B assay.Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL),and Fas gene transcription was determined by quantitative fluorescent PCR. RESULTS:The pcDNA3.1(+)/GPC3+α+EGFP plasmid was successfully constructed.In the experimental group,green fluorescence was observed at the cell periphery and in the cytoplasm,whereas in the negative control group,fluorescence was evenly distributed throughout the cell.Proliferation of the experimental group significantly decreased after 72 hours compared to the negative and blank control groups.Furthermore,the number of apoptotic cells was statistically different among the three groups as determined by a contingency table Chisquare test;the experimental group had the highest incidence of apoptosis.Fas gene transcription in the experimental group was higher than in the two control groups,and an increasing trend with time in the experimental group was observed. CONCLUSION:A chimeric,membrane-anchored protein, GPC3+α+EGFP,localized to the membrane of HepG2 cells and inhibited proliferation and accelerated apoptosis through a Fas-FasL pathway after co-cultivation with lymphocytes.展开更多
The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions.Previously,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from...The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions.Previously,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation.In order to fulfill the urgent requirement of thermostable lipolytic enzymes,in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate(pNP-C8)to p-nitrophenyl laurate(pNP-C12)by protein and solvent engineering.After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site,three variants with enhanced activity towards pNP-C12 were identified.Additionally,a combined mutant W474V/F488G/R526V/T560W was generated,which had the highest catalytic efficiency(kcat/Km)for pNP-C12,about 71-fold higher than the wild type.Its activity was further increased by solvent engineering,resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30%DMSO.The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation.It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center,which may facilitate binding and subsequent hydrolysis of bulky substrates.In conclusion,the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.展开更多
Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult...Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.展开更多
Chemical topology refers to the three-dimensional arrangement(i.e.,connectivity and spatial relationship)of a molecule's constituent atoms and bonds.The molecular mechanism for translation defines the linear confi...Chemical topology refers to the three-dimensional arrangement(i.e.,connectivity and spatial relationship)of a molecule's constituent atoms and bonds.The molecular mechanism for translation defines the linear configuration of all nascent proteins.Nontrivial protein topology arises only upon post-translational processing events and often imparts functional benefits such as enhanced stability,making topology a unique dimension for protein engineering.Utilizing the assembly-reaction synergy,our group has developed several methods for the effective and convenient cellular synthesis of a variety of topological proteins,such as lasso proteins,protein rotaxanes,and protein catenanes.The work opens the access to new protein classes and paves the road toward illustrating the topological effects on structure-function relationship of proteins,which lays solid foundation for exploring topological proteins’practical application.展开更多
Cell membrane integrity is fundamental to the normal activities of cells and is involved in both acute and chronic pathologies.Here,we report a probe for analyzing cell membrane integrity developed from a 9 nm-sized p...Cell membrane integrity is fundamental to the normal activities of cells and is involved in both acute and chronic pathologies.Here,we report a probe for analyzing cell membrane integrity developed from a 9 nm-sized protein nanocage named Dps via fluorophore conjugation with high spatial precision to avoid self-quenching.The probe cannot enter normal live cells but can accumulate in dead or live cells with damaged membranes,which,interestingly,leads to weak cytoplasmic and strong nuclear staining.This differential staining is found attributed to the high affinity of Dps for histones rather than DNA,providing a staining mechanism different from those of known membrane exclusion probes(MEPs).Moreover,the Dps nanoprobe is larger in size and thus applies a more stringent criterion for identifying severe membrane damage than currently available MEPs.This study shows the potential of Dps as a new bioimaging platform for biological and medical analyses.展开更多
8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,the availability of 8-PN is limited by inefficient prenyltransferases(PTs)and inadequate substrat...8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,the availability of 8-PN is limited by inefficient prenyltransferases(PTs)and inadequate substrate precursor levels in microbial chassis.First,six PTs from different sources and their truncated cognates were expressed in a(2S)-naringenin producing strain.Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate,tSfN8DT-1,could synthe-size 8-PN.Second,tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1^(Q12E)with greater catalytic activity was obtained.Third,key genes,tHMGR and IDI1,of the mevalonate(MVA)pathway were overexpressed using a copy number combinatorial strategy,which greatly improved 8-PN titer by 368.75%.Fourth,a predicted structure of tSfN8DT-1^(Q12E)was used for molecular docking and virtual saturation mutagenesis.Two key residues,P229 and N305,were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M.The best-performing mutant,tSfN8DT-1^(Q12EN305M),produced 49.35±0.05 mg/L(5.57±0.01 mg/g DCW)8-PN in a shaking flask.Finally,101.40±2.55 mg/L of 8-PN was obtained in a 5-L bioreactor,which is the greatest titer reported to date for 8-PN.This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1.The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods,which may provide a feasible strategy for the biosynthesis of prenylated flavonoids.展开更多
Rheumatoid arthritis(RA)is a relatively common inflammatory disease that affects the synovial tissue,eventually results in joints destruction and even long-term disability.Although Janus kinase inhibitors(Jakinibs)sho...Rheumatoid arthritis(RA)is a relatively common inflammatory disease that affects the synovial tissue,eventually results in joints destruction and even long-term disability.Although Janus kinase inhibitors(Jakinibs)show a rapid efficacy and are becoming the most successful agents in RA therapy,high dosing at frequent interval and severe toxicities cannot be avoided.Here,we developed a new type of fully compatible nanocarriers based on recombinant chimeric proteins with outstanding controlled release of upadacitinib.In addition,the fluorescent protein component of the nanocarriers enabled noninvasive fluorescence imaging of RA lesions,thus allowing real-time detection of RA therapy.Using rat models,the nanotherapeutic is shown to be superior to free upadacitinib,as indicated by extended circulation time and sustained bioefficacy.Strikingly,this nanosystem possesses an ultralong half-life of 45 h and a bioavailability of 4-times higher than pristine upadacitinib,thus extending the dosing interval from one day to 2 weeks.Side effects such as over-immunosuppression and leukocyte levels reduction were significantly mitigated.This smart strategy boosts efficacy,safety and visuality of Jakinibs in RA therapy,and potently enables customized designs of nanoplatforms for other therapeutics.展开更多
Vaccination is critical for population protection from pathogenic infections.However,its efficiency is frequently compromised by a failure of antigen retention and presentation.Herein,we designed a dextran-binding pro...Vaccination is critical for population protection from pathogenic infections.However,its efficiency is frequently compromised by a failure of antigen retention and presentation.Herein,we designed a dextran-binding protein DexBP,which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A,together with the sequence of the fluorescent protein mCherry.DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles.The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol)framework and a reactive oxygen species-responsive linker,obtaining magnetic polymeric microgels for carrying pathogen antigen.Similar to amoeba aggregation,the microgels self-assembled to form aggregates and further induced dendritic cell aggregation.This step-by-step assembly retained antigens at lymph nodes,promoted antigen presentation,stimulated humoral immunity,and protected the mice from lifethreatening systemic infections.This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections.展开更多
In this study, the modified hydrophobin, engineered for biomimetic mineralization, has been employed as a structure-directing agent for mineralization of calcium carbonate. For the first time amphiphilic calcium carbo...In this study, the modified hydrophobin, engineered for biomimetic mineralization, has been employed as a structure-directing agent for mineralization of calcium carbonate. For the first time amphiphilic calcium carbonate particles have been obtained, using engineered proteins. The mineral microparticles have been characterized by optical microscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD). While mineralization in the presence of non-modified hydrophobin results in polymorph mineral structures, uniform microspheres with an average particle diameter of one micron are obtained by employing hydrophobin which has been modified with an additional ceramophilic protein sequence. Owing to the tri-functionality of the modified hydrophobin (hydrophilic, hydrophobic and ceramophilic), the obtained mineral microparticles exhibit amphiphilic properties. Potential applications are in the areas of functional fillers and pigments, like biomedical and composite materials. Pickering emulsions have been prepared as a demonstration of the emulsion-stabilizing properties of the obtained amphiphilic mineral microspheres. The structure-directing effects of the studied engineered hydrophobins are compared with those of synthetic polymers (i.e. polycarboxylates) used as crystallization and scaling inhibitors in industrial applications.展开更多
Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster...Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.展开更多
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 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supported by China Postdoctoral Science Foundation Grant(2019M661742).
文摘Enhancing the stability of biomolecules is one of the hot topics in industry.In this study,we enhanced the stability of an important protein called LEPTIN.LEPTIN is a hormone secreted by fat cells playing an essential role in body weight and composition,and its deficiency can result in several disorders.The treatment of related LEPTIN dysfunctions is often available in the form of injection.To decrease the cost and the frequency of its applications can be achieved by increasing its lifetime through engineering LEPTIN.In this study,to engineer LEPTIN,we have introduced disulfide bonds.Disulfide By Design server was used to predict the suitable nominate pairs,which suggested three pairs of amino acids to be mutated to cysteine for disulfide bond formation.Additionally,to further evaluate the effect of combined mutations,we combined these three nominated pairs to produce three more mutants.In order to assess the effect of introduced mutations,molecular dynamic(MD)simulation was performed.The result suggests that Mutant-1 is more stable in comparison to wild-type and the other mutants.Moreover,docking results showed that the introduced mutation does not affect the receptor binding performance;therefore,it can be considered a suitable choice for future protein engineering.
基金Financial support from the National Natural Science Foundation of China(Grant Nos.52225001 and 51978485)the State Key Laboratory for Pollution Control(China)is acknowledged.
文摘The deep-learning protein structure prediction method AlphaFold2 has garnered enormous attention beyond the realm of structural biology,for its groundbreaking contribution to solving the"protein foiding problem"In this perspective,we explore the connection between protein structure studies and environmental research,delving into the potential for addressing specific environmental challenges.Proteins are promising for environmental applications because of the functional diversity endowed by their structural complexity.However,structural studies on proteins with environmental significance remain scarce.Here,we present the opportunity to study proteins by advancing experimental determination and deep-learning prediction methods.Specifically,the latest progress in environmental research via cryogenic electron microscopy is highlighted.It allows us to determine the structure of protein complexes in their native state within cells at molecular resolution,revealing environmentally-associated structural dynamics.With the remarkable advancements in computational power and experimental resolution,the study of protein structure and dynamics has reached unprecedented depth and accuracy.These advancements will undoubtedly accelerate the establishment of comprehensive environmental protein structural and functional databases.Tremendous opportunities for protein engineering exist to enable innovative solutions for environmental applications,such as the degradation of persistent contaminants,and the recovery of valuable metals as well as rare earth elements.
基金financially supported by the national first-class discipline program of Light Industry Technology and Engineering(LITE201820)the Key Technologies Research and Development Program of Jiangsu Province(BE2018623)。
文摘L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.
基金This work was supported by National Key Research and Development Program of China(2018YFA0901800 and 2018YFA0901400)National Natural Science Foundation of China(21978015,21636001,and 21776008).
文摘Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield and high cost.Microbial production represents as a promising alternative for efficient and sustainable production of PCs.In this review,we summarize recent advances in this field,which include enzyme mining and engineering to construct artificial pathways,balance of enzyme expression to improve pathway efficiency,coculture engineering to alleviate metabolic burden and side-reactions,and the use of genetic circuits for dynamic regulation and high throughput screening.Finally,current challenges and future perspectives for efficient production of PCs are also discussed.
基金supported by grants from the NaturalScience Foundation of China(30500239)the China PostdoctoralScience Foundation(20060400227)
文摘BACKGROUND:The incidence of hepatocellular carcinoma (HCC)in China is closely related to the population infected with hepatitis B virus(HBV).HCC cells with HBV secrete soluble HBsAg into blood but do not express it on the cell membrane This study aimed to construct and investigate a new glycosyl phosphatidylinositol(GPI)-anchored protein(GPC3+α+EGFP) as a DNA vaccine against HCC associated with HBV. METHODS:A recombinant plasmid(pcDNA3.1(+)/GPC3+ α+EGFP)was constructed and verified by restriction endo nuclease digestion and sequencing.pcDNA3.1(+)/GPC3+α+ EGFP was transfected into HepG2 cells(experimental group) using lipofectamine 2000.pEGFP-N1-transfected HepG2 cells were used as a negative control,and non-transfected HepG2 cells sreved as a blank control.HepG2 cells that steadily expressed the fusion protein GPC3+α+EGFP were screened by G418,propagated,and co-cultured with lymphocytes from healthy donors.Cell proliferation was measured by the classic sulforhodamine B assay.Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL),and Fas gene transcription was determined by quantitative fluorescent PCR. RESULTS:The pcDNA3.1(+)/GPC3+α+EGFP plasmid was successfully constructed.In the experimental group,green fluorescence was observed at the cell periphery and in the cytoplasm,whereas in the negative control group,fluorescence was evenly distributed throughout the cell.Proliferation of the experimental group significantly decreased after 72 hours compared to the negative and blank control groups.Furthermore,the number of apoptotic cells was statistically different among the three groups as determined by a contingency table Chisquare test;the experimental group had the highest incidence of apoptosis.Fas gene transcription in the experimental group was higher than in the two control groups,and an increasing trend with time in the experimental group was observed. CONCLUSION:A chimeric,membrane-anchored protein, GPC3+α+EGFP,localized to the membrane of HepG2 cells and inhibited proliferation and accelerated apoptosis through a Fas-FasL pathway after co-cultivation with lymphocytes.
基金supported by the National Basic Research Program of China(973 Program)the National Natural Science Foundation of China.
文摘The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions.Previously,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation.In order to fulfill the urgent requirement of thermostable lipolytic enzymes,in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate(pNP-C8)to p-nitrophenyl laurate(pNP-C12)by protein and solvent engineering.After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site,three variants with enhanced activity towards pNP-C12 were identified.Additionally,a combined mutant W474V/F488G/R526V/T560W was generated,which had the highest catalytic efficiency(kcat/Km)for pNP-C12,about 71-fold higher than the wild type.Its activity was further increased by solvent engineering,resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30%DMSO.The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation.It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center,which may facilitate binding and subsequent hydrolysis of bulky substrates.In conclusion,the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.
文摘Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.
基金We are grateful for the financial support from the National Key R&D Program of China(No.2020YFA0908100)the National Natural Science Foundation of China(Nos.21991132,21925102,92056118,22101010,22201016,22201017)Beijing National Laboratory for Molecular Sciences(BNLMS-CXXM-202006)。
文摘Chemical topology refers to the three-dimensional arrangement(i.e.,connectivity and spatial relationship)of a molecule's constituent atoms and bonds.The molecular mechanism for translation defines the linear configuration of all nascent proteins.Nontrivial protein topology arises only upon post-translational processing events and often imparts functional benefits such as enhanced stability,making topology a unique dimension for protein engineering.Utilizing the assembly-reaction synergy,our group has developed several methods for the effective and convenient cellular synthesis of a variety of topological proteins,such as lasso proteins,protein rotaxanes,and protein catenanes.The work opens the access to new protein classes and paves the road toward illustrating the topological effects on structure-function relationship of proteins,which lays solid foundation for exploring topological proteins’practical application.
基金This work was financially supported by the National Natural Science Foundation of China(No.31771103)Chinese Academy of Sciences(CAS)Emergency Project of African Swine Fever(ASF)Research(No.KJZD-SW-L07)the Scientific Instrument Developing Project of the CAS(No.YJKYYQ20190057).
文摘Cell membrane integrity is fundamental to the normal activities of cells and is involved in both acute and chronic pathologies.Here,we report a probe for analyzing cell membrane integrity developed from a 9 nm-sized protein nanocage named Dps via fluorophore conjugation with high spatial precision to avoid self-quenching.The probe cannot enter normal live cells but can accumulate in dead or live cells with damaged membranes,which,interestingly,leads to weak cytoplasmic and strong nuclear staining.This differential staining is found attributed to the high affinity of Dps for histones rather than DNA,providing a staining mechanism different from those of known membrane exclusion probes(MEPs).Moreover,the Dps nanoprobe is larger in size and thus applies a more stringent criterion for identifying severe membrane damage than currently available MEPs.This study shows the potential of Dps as a new bioimaging platform for biological and medical analyses.
基金supported by the National Key Research and Development Program of China(2019YFA0904800)the National Science Fund for Excellent Young Scholars(21822806)the National Natural Science Foundation of China(21908078).
文摘8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,the availability of 8-PN is limited by inefficient prenyltransferases(PTs)and inadequate substrate precursor levels in microbial chassis.First,six PTs from different sources and their truncated cognates were expressed in a(2S)-naringenin producing strain.Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate,tSfN8DT-1,could synthe-size 8-PN.Second,tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1^(Q12E)with greater catalytic activity was obtained.Third,key genes,tHMGR and IDI1,of the mevalonate(MVA)pathway were overexpressed using a copy number combinatorial strategy,which greatly improved 8-PN titer by 368.75%.Fourth,a predicted structure of tSfN8DT-1^(Q12E)was used for molecular docking and virtual saturation mutagenesis.Two key residues,P229 and N305,were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M.The best-performing mutant,tSfN8DT-1^(Q12EN305M),produced 49.35±0.05 mg/L(5.57±0.01 mg/g DCW)8-PN in a shaking flask.Finally,101.40±2.55 mg/L of 8-PN was obtained in a 5-L bioreactor,which is the greatest titer reported to date for 8-PN.This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1.The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods,which may provide a feasible strategy for the biosynthesis of prenylated flavonoids.
基金supported by the National Key R&D Program of China(Nos.2022YFA0913200,2021YFF0701800,2022YFF0710000,and 2020YFA0908900)the National Natural Science Foundation of China(Nos.22107097,22020102003,22277064,82272161,and 22125701)+1 种基金Tsinghua University Spring Breeze Fund grant(No.2021Z99CFZ005),and the Youth Innovation Promotion Association of CAS(No.2021226)All animal experiments were conducted in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of Chinawith the approval of the Institutional Animal Care and Use Committee of the Animal Experiment Center of Jilin University(No.PZPX20180929070).
文摘Rheumatoid arthritis(RA)is a relatively common inflammatory disease that affects the synovial tissue,eventually results in joints destruction and even long-term disability.Although Janus kinase inhibitors(Jakinibs)show a rapid efficacy and are becoming the most successful agents in RA therapy,high dosing at frequent interval and severe toxicities cannot be avoided.Here,we developed a new type of fully compatible nanocarriers based on recombinant chimeric proteins with outstanding controlled release of upadacitinib.In addition,the fluorescent protein component of the nanocarriers enabled noninvasive fluorescence imaging of RA lesions,thus allowing real-time detection of RA therapy.Using rat models,the nanotherapeutic is shown to be superior to free upadacitinib,as indicated by extended circulation time and sustained bioefficacy.Strikingly,this nanosystem possesses an ultralong half-life of 45 h and a bioavailability of 4-times higher than pristine upadacitinib,thus extending the dosing interval from one day to 2 weeks.Side effects such as over-immunosuppression and leukocyte levels reduction were significantly mitigated.This smart strategy boosts efficacy,safety and visuality of Jakinibs in RA therapy,and potently enables customized designs of nanoplatforms for other therapeutics.
基金This work was supported by the National Natural Science Foundation of China(Nos.3217010793 and 31870139)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(No.TSBICIP-KJGG-006)+1 种基金the Natural Science Foundation of Tianjin(No.19JCZDJC33800)the Fundamental Research Funds for the Central Universities.
文摘Vaccination is critical for population protection from pathogenic infections.However,its efficiency is frequently compromised by a failure of antigen retention and presentation.Herein,we designed a dextran-binding protein DexBP,which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A,together with the sequence of the fluorescent protein mCherry.DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles.The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol)framework and a reactive oxygen species-responsive linker,obtaining magnetic polymeric microgels for carrying pathogen antigen.Similar to amoeba aggregation,the microgels self-assembled to form aggregates and further induced dendritic cell aggregation.This step-by-step assembly retained antigens at lymph nodes,promoted antigen presentation,stimulated humoral immunity,and protected the mice from lifethreatening systemic infections.This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections.
基金Funding is gratefully acknowledged by VTT’s Frontier Project Biomimetic Hybrid Materials.
文摘In this study, the modified hydrophobin, engineered for biomimetic mineralization, has been employed as a structure-directing agent for mineralization of calcium carbonate. For the first time amphiphilic calcium carbonate particles have been obtained, using engineered proteins. The mineral microparticles have been characterized by optical microscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD). While mineralization in the presence of non-modified hydrophobin results in polymorph mineral structures, uniform microspheres with an average particle diameter of one micron are obtained by employing hydrophobin which has been modified with an additional ceramophilic protein sequence. Owing to the tri-functionality of the modified hydrophobin (hydrophilic, hydrophobic and ceramophilic), the obtained mineral microparticles exhibit amphiphilic properties. Potential applications are in the areas of functional fillers and pigments, like biomedical and composite materials. Pickering emulsions have been prepared as a demonstration of the emulsion-stabilizing properties of the obtained amphiphilic mineral microspheres. The structure-directing effects of the studied engineered hydrophobins are compared with those of synthetic polymers (i.e. polycarboxylates) used as crystallization and scaling inhibitors in industrial applications.
文摘Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.
