Directed Evolution of L-Aspartase by Mobility of Domains

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.

Directed Evolution of Human Cu,Zn-SOD Under Artificial Oxidant Pressures

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.

Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae

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.

Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde

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.

Advances in the study of directed evolution for cellulases

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.

Biochemical characterization of a polyethylene terephthalate hydrolase and design of high-throughput screening for its directed evolution

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.

Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes

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.

Directed Evolution of Insoluble Arabidopsis thaliana Zeta Class Glutathione S-Transferase Mutants for Higher Solubility in Escherichia coli

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）.

Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

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.

Creation of cytochrome P450 catalysis depending on a non-natural cofactor for fatty acid hydroxylation

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.

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

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.

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

Naturally occurring structural variations(SVs)are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes.Controllable methods aimed at introducing the complex SVs and their related molecular mechanisms have remained farfetched.In this study,an SV-prone yeast strain was developed using Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution(SCRaMbLE)technology with two synthetic chromosomes,namely synV and synX.The biosynthesis of astaxanthin is used as a readout and a proof of concept for the application of SVs in industries.Our findings showed that complex SVs,including a pericentric inversion and a trans-chromosome translocation between synV and synX,resulted in two neo-chromosomes and a 2.7-fold yield of astaxanthin.Also,genetic targets were mapped,which resulted in a higher astaxanthin yield,thus demonstrating the SVs’ability to reorganize genetic information along the chromosomes.The rational design of trans-chromosome translocation and pericentric inversion enabled precise induction of these phenomena.Collectively,this study provides an effective tool to not only accelerate the directed genome evolution but also to reveal the mechanistic insight of complex SVs for altering phenotypes.

Topology Optimization of Self-Supporting Structures for Additive Manufacturing with Adaptive Explicit Continuous Constraint

The integration of topology optimization(TO)and additive manufacturing(AM)technologies can create significant synergy benefits,while the lack of AM-friendly TO algorithms is a serious bottleneck for the application of TO in AM.In this paper,a TO method is proposed to design self-supporting structures with an explicit continuous self-supporting constraint,which can be adaptively activated and tightened during the optimization procedure.The TO procedure is suitable for various critical overhang angles(COA),which is integrated with build direction assignment to reduce performance loss.Besides,a triangular directional self-supporting constraint sensitivity filter is devised to promote the downward evolution of structures and maintain stability.Two numerical examples are presented;all the test cases have successfully converged and the optimized solutions demonstrate good manufacturability.In the meanwhile,a fully self-supporting design can be obtained with a slight cost in performance through combination with build direction assignment.

Low-thrust trajectory optimization for multiple target bodies tour mission

Spacecraft science missions to planets or asteroids have historically visited only one or several celestial bodies per mission.The research goal of this paper is to create a trajectory design algorithm that generates trajectory allowing a spacecraft to visit a significant number of asteroids during a single mission.For the problem of global trajectory optimization,even with recent advances in low-thrust trajectory optimization,a full enumeration of this problem is not possible.This work presents an algorithm to traverse the searching space in a practical fashion and generate solutions.The flight sequence is determined in ballistic scenario,and a differential evolution method is used with constructing a three-impulse transfer problem,then the local optimization is implemented with low-thrust propulsion on the basis of the solutions of impulsive trajectories.The proposed method enables trajectory design for multiple asteroids tour by using available low thrust propulsion technology within fuel and time duration constraints.

Continuous-flow synthesis of polysubstituted γ-butyrolactones via enzymatic cascade catalysis

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.

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving pho- tosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an effi- cient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coil-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G sub- stitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxyla- tion activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

Asymmetric synthesis of syn-aryl-(2S,3R)-2-chloro-3-hydroxy esters via an engineered ketoreductase-catalyzed dynamic reductive kinetic resolution

We report here a generic,green synthesis of 17 valuable syn-aryl-(2S,3R)-2–chloro-3–hydroxy esters(syn-(2S,3R)-1)in 73%-99%isolated yields along with 6.1:1–83:1 dr and 31%~>99%ee,through dynamic reductive kinetic resolution of racemic arylα–chloroβ-keto esters(2)catalyzed by an engineered ketoreductase which was obtained via ep PCR-based directed evolution.The hectogram scale synthesis of syn-(2S,3R)-1b at a substrate concentration of 120 g/L showcased the application potential of the biocatalytic method developed presently.

Book review of“Enzyme engineering:Selective catalysts for applications in biotechnology,organic chemistry,and life science”

This book[1]begins with an introductory chapter in which the history of directed enzyme evolution is briefly presented and the different gene mutagenesis techniques are outlined,followed by a comprehensive chapter describing medium-and high-throughput screening systems for assaying stereoselectivity and activity.

Development of a dual temperature control system for isoprene biosynthesis in Saccharomyces cerevisiae

Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter PSSA4 to control the expression of PGAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

Glycoenzyme Tool Development:Principles,Screening Methods,and Recent Advances

To elucidate the relationship between carbohydrate structure and its biological function,glycoenzyme tools are widely applied in a variety of physio-logical and pathological scenarios.Intricate adjustment via glycoenzyme engineering improved the catalytic efficiency,extended the repertoire of accessible catalytic reactions,and tailored novel substrate specificities.In this review,we introduce the principles of glycoenzyme engineering and summarize the recent advances in the rational design and directed evolution of these enzymes,with an emphasis on screening methods in directed evolution.We also excerpt novel detection methods in glycobiology that can be adapted for the development of next-generation glycoenzyme tools.