In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by ...In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.展开更多
To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetram...To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_ cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.展开更多
Cu,Zn SOD is a highly conserved enzyme and the controversy about its evolutionary possibility in the near future has been lively. In order to further our understanding of the future fate of human Cu,Zn SOD, we adopt...Cu,Zn SOD is a highly conserved enzyme and the controversy about its evolutionary possibility in the near future has been lively. In order to further our understanding of the future fate of human Cu,Zn SOD, we adopted a strategy relating to the directed evolution to study how the mutants of human Cu,Zn SOD respond to different oxidative stress. After five rounds of screening, we found a mutant that can survive under harsh pressures and DNA sequencing proves that it shows a mutation responsible for the phenomenon. However, under natural pressure, our screening comes to nothing. Then we may draw the following conclusions: the evolution of biological macromolecules in some respect depends on their surroundings and if they are too familiar with a certain environment, they may embody evolutionary inertia.展开更多
Expression of recombinant protein in Escherichia coli （E.coli） is generally considered as one of the ideal systems to produce proteins for industrial production.However,the majority of proteins usually fail to fold ...Expression of recombinant protein in Escherichia coli （E.coli） is generally considered as one of the ideal systems to produce proteins for industrial production.However,the majority of proteins usually fail to fold into their native state and accumulate as insoluble inclusion bodies with no biological activity in E.coli（Yang et al.,2003）.展开更多
Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide ad...Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(MDH)catalyzes the oxidation of methanol to formaldehyde via NADH generation,providing a highly active C1 intermediate and reducing power for subsequent biosynthesis.However,the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization,especially in nicotinamide adenine dinucleotide phosphate(NADP^(+))-dependent biosynthesis.Herein,we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334.NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs,establishing a positive correlation between the cell growth rate and MDH activity.Using this principle,MDH mutants exhibiting a 20-fold improvement in catalytic efficiency(k_(cat)/K_(m))and a 90-fold cofactor specificity switch from NAD^(+)to NADP+without a decrease in specific enzyme activity,were efficiently screened from random and semi-rationally designed libraries.We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)^(+)-dependent enzymes.展开更多
catalyzed byβ-carotene hydroxylase(crtZ)andβ-carotene ketolase(crtW)decreases the content of the astaxanthin.Here,we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis...catalyzed byβ-carotene hydroxylase(crtZ)andβ-carotene ketolase(crtW)decreases the content of the astaxanthin.Here,we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae.The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin.Compared with the control strain,the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times,respectively.Moreover,9 variant fusion mutants with improved astaxanthin production were generated through directed evolution.Combining these dominant mutants generated a variant,L95S+I206L,which increased the astaxanthin content of 3.8 times than the control strain.The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket.Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.展开更多
If cellulose can be effectively hydrolyzed intoglucose by cellulase,the production costs of hydrogen,ethanol or other chemicals from cellulosic materials will begreatly decreased,and economically viable production ofb...If cellulose can be effectively hydrolyzed intoglucose by cellulase,the production costs of hydrogen,ethanol or other chemicals from cellulosic materials will begreatly decreased,and economically viable production ofbiohydrogen and bioethanol will become feasible.Celluloseis degraded into glucoses by multi-component enzymesystems.Nowadays cellulases are widely used in brewing,food,bioenergy,fodder,textiles,paper,pharmaceuticals,environmental protection and other industries.However,existing cellulases have several problems that limit theirwider applications,including the low turnover number forsolid cellulosic materials,and low stability in adapting tovarious application conditions.For example,high temperature,low pH,and so on.Application of directedevolution technology may be one of the most effectiveways for improving the characteristics of cellulases.Thispaper presents a brief review of the cellulases hydrolysismechanism by cellulase,advances in cellulases(endoglucanaseandβ-glucosidase)improvement by directedevolution for several characteristics(for instance,thermalstability,pH adaptability and enzyme activity),limitationsof directed evolution for cellulases,and the outlook fordirected evolution for cellulase.展开更多
Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic...Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to formaldehyde.Herein,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic activity.The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants.Mdh_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation libraries.The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity.The beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-helices.Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.展开更多
Objective To devellop directly molecular evolution Of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremelly slow and are unable to nitrify effectively inorganic nitrogen in wastew...Objective To devellop directly molecular evolution Of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremelly slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatmem. Methods The norB gene coding the ntitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PeR and staggered extension process PCR, the sequence was differem from its parental DNA fragmems and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L.d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures. Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.展开更多
d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential sweeteners.At present,the main production method of d-allulose is epimerization...d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential sweeteners.At present,the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase(DAE).However,industrial applications of DAE are still limited by its poor thermostability.Herein,directed evolution was applied to improve the thermostability of dAE from Clostridium cellulolyticum H10(CcDAE).Two optimal mutants D281G and C289R,exhibiting 13.80-fold and 13.88-fold t_(1/2 )values as that of wild type at 65℃,respectively,were obtained.To further enhance the thermostability,the triple mutant A107P/D281G/C289R was constructed after combina-tion of mutants D281G,C289R,and previously identified thermostability-enhanced mutant A107P.The T_(m) and optimal temperature of triple mutant were increased by 14.39℃and 5℃,respectively,compared to the wild type,meanwhile,the half-life of triple mutant was 58.85-fold as that of wild type at 65℃.Furthermore,the conversion rate of triple mutant was increased from 24.76%of wild type to 27.53%using 300 g/L d-fructose as substrate at 70℃.The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.展开更多
Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attr...Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.展开更多
Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-p...Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-proteins-for-multi-tasks"NE in terms of screening throughput and efficiency,thus often failing to meet the fast-growing demands for biocatalysts with desired properties.In this study,we design a novel"multi-enzymes-for-multi-substrates"(MEMS)DE model and establish the proof-ofconcept by running a NE-mimicking and higher-throughput screening on the basis of"two-P450 s-against-seven-substrates"(2P×7S)in one pot.With the multiplied throughput and improved hit rate,we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes(P450 BM3 and P450 cam)in laboratory.It is anticipated that the new strategy of MEMS DE will find broader application for a larger repertoire of enzymes in the future.Furthermore,structural and substrate docking analysis of the two functionally convergent P450 variants provide important insights into how distinct P450 active-sites can reach a common catalytic goal.展开更多
The enzymatic depolymerization of polyethylene terephthalate(PET)offers a sustainable approach for the recycling of PET waste.Great efforts have been devoted to engineering PET depolymerases on the substrate binding c...The enzymatic depolymerization of polyethylene terephthalate(PET)offers a sustainable approach for the recycling of PET waste.Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface.Here,we report the systematic engineering of whole β-sheet regions in the core of IsPETase(a PETase from Ideonella sakaiensis)via a fluorescent high-throughput screening assay.Twenty-one beneficial substitutions were obtained and iteratively recombined.The best variant,DepoPETase β,with an increase in the melting temperatures(T_(m))of 22.9℃,exhibited superior depolymerization performance and enabled complete depolymerization of100.5 g of untreated post-consumer PET(pc-PET;0.26% W_(enzyme)/W_(PET) enzyme loading)in liter-scale bioreactor at 50℃within 4 d.Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region,a more tightly packed structure of the core sheets and the surrounding helix,and improved binding of PET to the active sites.This study not only demonstrates the importance of engineering strategy in theβ-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.展开更多
Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In ...Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In this study,IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide(antiepileptic drug Brivaracetam)via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and(S)-2-aminobutylamide with high diastereoselectivity.To further improve the catalytic efficiency of IR104,its mutant D191E/L195I/E253S/M258A(M3)was identified by saturation mutagenesis and iterative combinatorial mutagenesis,which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity(98:2 d.r.).Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme.The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemicα-substituted aldehydo-esters,expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination.Finally,IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale.Using this mutant,various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r.and 96%yields and subsequently converted intoγ-andδ-lactams,providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.展开更多
We use the directional slacks-based measure of efficiency and inverse distance weighting method to analyze the spatial pattern evolution of the industrial green total factor productivity of 108 cities in the Yangtze R...We use the directional slacks-based measure of efficiency and inverse distance weighting method to analyze the spatial pattern evolution of the industrial green total factor productivity of 108 cities in the Yangtze River Economic Belt in 2003–2013.Results show that both the subprime mortgage crisis and ‘the new normal' had significant negative effects on productivity growth,leading to the different spatial patterns between 2003–2008 and 2009–2013.Before 2008,green poles had gathered around some capital cities and formed a tripartite pattern,which was a typical core-periphery pattern.Due to a combination of the polarization and the diffusion effects,capital cities became the growth poles and ‘core' regions,while surrounding areas became the ‘periphery'.This was mainly caused by the innate advantage of capital cities and ‘the rise of central China' strategy.After 2008,the tripartite pattern changed to a multi-poles pattern where green poles continuously and densely spread in the midstream and downstream areas.This is due to the regional difference in the leading effect of green poles.The leading effect of green poles in midstream and downstream areas has changed from polarization to diffusion,while the polarization effect still leads in the upstream area.展开更多
Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis o...Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis of these molecules faces the problems of low enzyme activity and phase separation in batch reaction, resulting in low productivity. Herein, we report a new continuous-flow process to synthesize the optically pure Nicotiana tabacum lactone(3S,4S)-4a and whisky lactone(3R,4S)-4b from α,β-unsaturatedγ-ketoesters. A new ene reductase(ER) from Swingsia samuiensi(Ss ER) and a carbonyl reductase(Ss CR)were engineered by directed evolution to improve their activity and thermostability. The continuous-flow preparative reactions were performed in two 3D microfluidic reactors, generating(3S,4S)-4a(99% ee and87% de) and(3R,4S)-4b(99% ee and 98% de) with space-time yields 3 and 7.4 times higher than those of the batch reactions. The significant enhancement in the productivity of enzyme cascade catalysis brought by cutting-edge continuous microfluidic technology will benefit the general multi-enzyme catalytic systems in the future.展开更多
Cytidine triphosphate(CTP),as a substance involved in the metabolism of phospholipids,proteins and nucleic acids,has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline.In this...Cytidine triphosphate(CTP),as a substance involved in the metabolism of phospholipids,proteins and nucleic acids,has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline.In this study,we established an in vitro six-enzyme cascade system to generate CTP.To avoid thermodynamic bottlenecks,we employed a circuitous and two-stage reaction strategy.Using cytidine as the key substrate,the final product CTP is obtained via the deamination and uridine phosphorylation pathways,relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate.Several extremophilic microbial-derived deaminases were screened and characterized,and a suitable cytidine deaminase was selected to match the first-stage reaction conditions.In addition,directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition,along with a 1.7-fold increase in activity over the wild type.After optimizing the reaction conditions,we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM,and the yield of CTP exceeded 82%within 10.0 h.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
Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-p...Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.展开更多
To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a ...To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.展开更多
基金Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of ZhejiangProvince (No. Z304076), China
文摘In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.
文摘To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_ cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes.
文摘Cu,Zn SOD is a highly conserved enzyme and the controversy about its evolutionary possibility in the near future has been lively. In order to further our understanding of the future fate of human Cu,Zn SOD, we adopted a strategy relating to the directed evolution to study how the mutants of human Cu,Zn SOD respond to different oxidative stress. After five rounds of screening, we found a mutant that can survive under harsh pressures and DNA sequencing proves that it shows a mutation responsible for the phenomenon. However, under natural pressure, our screening comes to nothing. Then we may draw the following conclusions: the evolution of biological macromolecules in some respect depends on their surroundings and if they are too familiar with a certain environment, they may embody evolutionary inertia.
基金supported by the grants of the National Natural Science Foundation of China(No.31070717)Tianjin International Science and Technology Cooperation Project (No.09ZCGHHZ00500)the 111 Project(No.B08011)
文摘Expression of recombinant protein in Escherichia coli （E.coli） is generally considered as one of the ideal systems to produce proteins for industrial production.However,the majority of proteins usually fail to fold into their native state and accumulate as insoluble inclusion bodies with no biological activity in E.coli（Yang et al.,2003）.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110201)the National Key R&D Program of China(2018YFA0901500)+3 种基金the National Natural Science Foundation of China(32070083 and 32222004)the Innovation Fund of Haihe Laboratory of Synthetic Biology(22HHSWSS00017)the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008).
文摘Methanol,produced from carbon dioxide,natural gas,and biomass,has drawn increasing attention as a promising green carbon feedstock for biomanufacturing due to its sustainable and energy-rich properties.Nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(MDH)catalyzes the oxidation of methanol to formaldehyde via NADH generation,providing a highly active C1 intermediate and reducing power for subsequent biosynthesis.However,the unsatisfactory catalytic efficiency and cofactor bias of MDH significantly impede methanol valorization,especially in nicotinamide adenine dinucleotide phosphate(NADP^(+))-dependent biosynthesis.Herein,we employed synthetic NADH and NADPH auxotrophic Escherichia coli strains as growth-coupled selection platforms for the directed evolution of MDH from Bacillus stearothermophilus DSM 2334.NADH or NADPH generated by MDH-catalyzed methanol oxidation enabled the growth of synthetic cofactor auxotrophs,establishing a positive correlation between the cell growth rate and MDH activity.Using this principle,MDH mutants exhibiting a 20-fold improvement in catalytic efficiency(k_(cat)/K_(m))and a 90-fold cofactor specificity switch from NAD^(+)to NADP+without a decrease in specific enzyme activity,were efficiently screened from random and semi-rationally designed libraries.We envision that these mutants will advance methanol valorization and that the synthetic cofactor auxotrophs will serve as versatile selection platforms for the evolution of NAD(P)^(+)-dependent enzymes.
基金funded by the Ministry of Science and Technology,the National Key Research and Development Program of China (2021YFC2100800)the National Natural Science Foundation of China (31800719,and 21621004).
文摘catalyzed byβ-carotene hydroxylase(crtZ)andβ-carotene ketolase(crtW)decreases the content of the astaxanthin.Here,we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae.The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin.Compared with the control strain,the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times,respectively.Moreover,9 variant fusion mutants with improved astaxanthin production were generated through directed evolution.Combining these dominant mutants generated a variant,L95S+I206L,which increased the astaxanthin content of 3.8 times than the control strain.The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket.Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.
基金This research was supported by the National Natural Science Foundation of China(Grant No.30870037)Research Fund for the Doctoral Program of Higher Education of China(No.20102329120002)China Postdoctoral Science Foundation(No.20090450983).
文摘If cellulose can be effectively hydrolyzed intoglucose by cellulase,the production costs of hydrogen,ethanol or other chemicals from cellulosic materials will begreatly decreased,and economically viable production ofbiohydrogen and bioethanol will become feasible.Celluloseis degraded into glucoses by multi-component enzymesystems.Nowadays cellulases are widely used in brewing,food,bioenergy,fodder,textiles,paper,pharmaceuticals,environmental protection and other industries.However,existing cellulases have several problems that limit theirwider applications,including the low turnover number forsolid cellulosic materials,and low stability in adapting tovarious application conditions.For example,high temperature,low pH,and so on.Application of directedevolution technology may be one of the most effectiveways for improving the characteristics of cellulases.Thispaper presents a brief review of the cellulases hydrolysismechanism by cellulase,advances in cellulases(endoglucanaseandβ-glucosidase)improvement by directedevolution for several characteristics(for instance,thermalstability,pH adaptability and enzyme activity),limitationsof directed evolution for cellulases,and the outlook fordirected evolution for cellulase.
基金the National Key Research and Development Program of China(2018YFA0901500)the National Natural Science Foundation of China(32070083 and 32222004)+2 种基金the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-008)the Innovation Fund of Haihe Laboratory of Synthetic Biology.
文摘Methanol is a promising one-carbon feedstock for biomanufacturing,which can be sustainably produced from carbon dioxide and natural gas.However,the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide(NAD^(+))-dependent methanol dehydrogenase(Mdh)that oxidizes methanol to formaldehyde.Herein,the neutrophilic and mesophilic NAD^(+)-dependent Mdh from Bacillus stearothermophilus DSM 2334(Mdh_(Bs))was subjected to directed evolution for enhancing the catalytic activity.The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants.Mdh_(Bs)variants with up to 6.5-fold higher K_(cat)/K_(M)value for methanol were screened from random mutation libraries.The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity.The beneficial T153P mutation changes the interaction network of this residue and breaks theα-helix important for substrate binding into two shortα-helices.Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve Mdh_(Bs),and this study provides an efficient strategy for directed evolution of Mdh.
基金This study was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2001AA214191).
文摘Objective To devellop directly molecular evolution Of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremelly slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatmem. Methods The norB gene coding the ntitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PeR and staggered extension process PCR, the sequence was differem from its parental DNA fragmems and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L.d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures. Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.
基金The authors are grateful for the financial support from the National Natural Science Foundation of China(NSFC)(32101884)Natural Science Foundation of Jiangsu Province(BK20190586).
文摘d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential sweeteners.At present,the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase(DAE).However,industrial applications of DAE are still limited by its poor thermostability.Herein,directed evolution was applied to improve the thermostability of dAE from Clostridium cellulolyticum H10(CcDAE).Two optimal mutants D281G and C289R,exhibiting 13.80-fold and 13.88-fold t_(1/2 )values as that of wild type at 65℃,respectively,were obtained.To further enhance the thermostability,the triple mutant A107P/D281G/C289R was constructed after combina-tion of mutants D281G,C289R,and previously identified thermostability-enhanced mutant A107P.The T_(m) and optimal temperature of triple mutant were increased by 14.39℃and 5℃,respectively,compared to the wild type,meanwhile,the half-life of triple mutant was 58.85-fold as that of wild type at 65℃.Furthermore,the conversion rate of triple mutant was increased from 24.76%of wild type to 27.53%using 300 g/L d-fructose as substrate at 70℃.The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.
基金supported by the Qilu Youth Scholar Startup Funding of Shandong University(L.H.)National Natural Science Foundation of China(32170038)as well as the Sino-German mobility programme(M-0348).
文摘Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.
基金supported by the National Key Research and Development Program of China(2019YFA0706900)the National Natural Science Foundation of China(32025001,31872729,31600045,32071266,31800664,82022066,and 31800041)+5 种基金the Natural Science Foundation of Shandong Province,China(ZR2019ZD20,ZR2016CQ05,and ZR2019QC009)the Laboratory for Marine Drugs and Bioproducts of Pilot National Laboratory for Marine Science and Technology(Qingdao)(LMDBKF-2019-01)the Tianjin Synthetic Biotechnology Innovation Capability Improvement Project(TSBICIP-KJGG-001)the State Key Laboratory of Bio-organic and Natural Products Chemistry(SKLBNPC18242)the Fundamental Research Funds of Shandong University(2019GN030 and 2019GN033)the Foundation of Qilu University of Technology of Cultivating Subject for Biology and Biochemistry(No.202014)。
文摘Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-proteins-for-multi-tasks"NE in terms of screening throughput and efficiency,thus often failing to meet the fast-growing demands for biocatalysts with desired properties.In this study,we design a novel"multi-enzymes-for-multi-substrates"(MEMS)DE model and establish the proof-ofconcept by running a NE-mimicking and higher-throughput screening on the basis of"two-P450 s-against-seven-substrates"(2P×7S)in one pot.With the multiplied throughput and improved hit rate,we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes(P450 BM3 and P450 cam)in laboratory.It is anticipated that the new strategy of MEMS DE will find broader application for a larger repertoire of enzymes in the future.Furthermore,structural and substrate docking analysis of the two functionally convergent P450 variants provide important insights into how distinct P450 active-sites can reach a common catalytic goal.
基金funded by the National Key Research and Development Program of China(2023YFC3903300)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIPIJCP-003,TSBICIP-KJGG-009-0203,and TSBICIP-BRFI-005)the Innovation Fund of Haihe Laboratory of Synthetic Biology(22HHSWSS00018)。
文摘The enzymatic depolymerization of polyethylene terephthalate(PET)offers a sustainable approach for the recycling of PET waste.Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface.Here,we report the systematic engineering of whole β-sheet regions in the core of IsPETase(a PETase from Ideonella sakaiensis)via a fluorescent high-throughput screening assay.Twenty-one beneficial substitutions were obtained and iteratively recombined.The best variant,DepoPETase β,with an increase in the melting temperatures(T_(m))of 22.9℃,exhibited superior depolymerization performance and enabled complete depolymerization of100.5 g of untreated post-consumer PET(pc-PET;0.26% W_(enzyme)/W_(PET) enzyme loading)in liter-scale bioreactor at 50℃within 4 d.Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region,a more tightly packed structure of the core sheets and the surrounding helix,and improved binding of PET to the active sites.This study not only demonstrates the importance of engineering strategy in theβ-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.
文摘Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In this study,IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide(antiepileptic drug Brivaracetam)via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and(S)-2-aminobutylamide with high diastereoselectivity.To further improve the catalytic efficiency of IR104,its mutant D191E/L195I/E253S/M258A(M3)was identified by saturation mutagenesis and iterative combinatorial mutagenesis,which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity(98:2 d.r.).Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme.The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemicα-substituted aldehydo-esters,expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination.Finally,IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale.Using this mutant,various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r.and 96%yields and subsequently converted intoγ-andδ-lactams,providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.
基金Under the auspices of the post-funded project of National Social Science Foundation of China(No.16FJL009)
文摘We use the directional slacks-based measure of efficiency and inverse distance weighting method to analyze the spatial pattern evolution of the industrial green total factor productivity of 108 cities in the Yangtze River Economic Belt in 2003–2013.Results show that both the subprime mortgage crisis and ‘the new normal' had significant negative effects on productivity growth,leading to the different spatial patterns between 2003–2008 and 2009–2013.Before 2008,green poles had gathered around some capital cities and formed a tripartite pattern,which was a typical core-periphery pattern.Due to a combination of the polarization and the diffusion effects,capital cities became the growth poles and ‘core' regions,while surrounding areas became the ‘periphery'.This was mainly caused by the innate advantage of capital cities and ‘the rise of central China' strategy.After 2008,the tripartite pattern changed to a multi-poles pattern where green poles continuously and densely spread in the midstream and downstream areas.This is due to the regional difference in the leading effect of green poles.The leading effect of green poles in midstream and downstream areas has changed from polarization to diffusion,while the polarization effect still leads in the upstream area.
基金financially sponsored by the National Key Research and Development Program of China (No.2021YFC2102804)the National Natural Science Foundation of China(No.22078096)。
文摘Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis of these molecules faces the problems of low enzyme activity and phase separation in batch reaction, resulting in low productivity. Herein, we report a new continuous-flow process to synthesize the optically pure Nicotiana tabacum lactone(3S,4S)-4a and whisky lactone(3R,4S)-4b from α,β-unsaturatedγ-ketoesters. A new ene reductase(ER) from Swingsia samuiensi(Ss ER) and a carbonyl reductase(Ss CR)were engineered by directed evolution to improve their activity and thermostability. The continuous-flow preparative reactions were performed in two 3D microfluidic reactors, generating(3S,4S)-4a(99% ee and87% de) and(3R,4S)-4b(99% ee and 98% de) with space-time yields 3 and 7.4 times higher than those of the batch reactions. The significant enhancement in the productivity of enzyme cascade catalysis brought by cutting-edge continuous microfluidic technology will benefit the general multi-enzyme catalytic systems in the future.
基金supported by the National Key Research and Development Program of China(2022YFC3401700)the National Natural Science Foundation of China(Grant No.32171478)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(Grant No.TSBICIP-KJGG-009).
文摘Cytidine triphosphate(CTP),as a substance involved in the metabolism of phospholipids,proteins and nucleic acids,has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline.In this study,we established an in vitro six-enzyme cascade system to generate CTP.To avoid thermodynamic bottlenecks,we employed a circuitous and two-stage reaction strategy.Using cytidine as the key substrate,the final product CTP is obtained via the deamination and uridine phosphorylation pathways,relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate.Several extremophilic microbial-derived deaminases were screened and characterized,and a suitable cytidine deaminase was selected to match the first-stage reaction conditions.In addition,directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition,along with a 1.7-fold increase in activity over the wild type.After optimizing the reaction conditions,we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM,and the yield of CTP exceeded 82%within 10.0 h.
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 Key R&D Program of China(2019YFA0904900)the National Natural Science Foundation of China(21877112,21837002,21721004)。
文摘Cytochrome P450 enzymes catalyze diverse oxidative transformations at the expense of reduced nicotinamide adenine dinucleotide phosphate(NADPH),however,their applications remain limited largely because NADPH is cost-prohibitive for biocatalysis at scale yet tightly regulated in host cells.A highly challenging task for P450 catalysis has been to develop an alternative and biocompatible electrondonating system.Here we engineered P450 BM3 to favor reduced nicotinamide cytosine dinucleotide(NCDH)and created non-natural cofactor-dependent P450 catalysis.Two outstanding mutants were identified with over 640-fold NCDH preference improvement and good catalytic efficiencies of over15,000 M^(-1)s^(-1)for the oxidation of the fatty acid probe 12-(para-nitrophenoxy)-dodecanoate.Molecular docking analysis indicated that these mutants bear a compacted cofactor entrance.Upon fusing with an NCD-dependent formate dehydrogenase,fused proteins functioned as NCDH-specific P450catalysts by using formate as the electron donor.Importantly,these mutants and fusions catalyzed NCDH-dependent hydroxylation of fatty acids with similar chain length preference to those by natural P450 BM3 in the presence of NADPH and also similar regioselectivity for subterminal hydroxylation of lauric acid.As P450 BM3 and its variants are catalytically powerful to take diverse substrates and convey different reaction paths,our results offer an exciting opportunity to devise advanced cell factories that convey oxidative biocatalysis with an orthogonal reducing power supply system.
基金Supported by the National Natural Science Foundation of China(Nos30400081, 30570405 and 20672045)the Key Tech-nology Research and Development Program of China(No2004BA713D03-04)
文摘To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.
