Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.     

Enhanced enantioselectivity of a carboxyl esterase from Rhodobacter sphaeroides by directed evolution

The present work created an esterase variant from Rhodobacter sphaeroides (RspE) with enhanced selectivity in hydrolytic kinetic resolutions by directed evolution. A “model” substrate, methyl mandelate, was introduced in the high-throughput screening procedure. E values of a variant CH (Asn62Cys/Leu145His) for six different esters were 10–83, which were a relative improvement compared to 2–20 for the wild type. Our subsequent crystal structure interpretation and molecular dynamics simulations helped shed light on the source of enantioselectivity modified by directed evolution. Though mutations displayed no “direct” interaction with the substrate, they were hypothesized to strengthen the intramolecular interaction in the catalytic cavity of variant. Conformation analysis revealed that the enhanced enantioselectivity of variant CH for the seven substrates applied in this study was derived from the decrease in size of the substrate binding pocket.     

Directed evolution of metagenome-derived epoxide hydrolase for improved enantioselectivity and enantioconvergence

We performed a directed evolution study with a metagenome-derived epoxide hydrolase (EH), termed Kau2. Homology models of Kau2 were built; we selected one of them and used it as a guide for saturation mutagenesis experiments targeted at specific residues within the large substrate binding pocket. During the molecular evolution process, we found several enzyme variants with higher enantioselectivity or enhanced enantioconvergence toward para-Chlorostyrene oxide. Improved enantioselectivities by up to a factor of 5, reaching an E-value of up to 130 with the R-enantiomer as the residual epoxide, were achieved by replacing amino acid pairs at the positions 110 and 113, or 290 and 291, which are positions located in the vicinity of two presumed binding sites for the epoxide enantiomers. The (R)-para-Chlorophenylethane-1,2-diol product exhibited a high enantiomeric excess (ee) of 97% at 50% conversion of the racemic epoxide for the most enantioselective variant. Further, five amino acid substitutions were sufficient to substantially increase the degree of enantioconvergence and to lower the E-value to 17 for the final evolved EH variant, enabling the production of the R-diol with an ee-value of 93% at 28 °C in a complete conversion of the racemic epoxide. Higher ee_p-values of up to 97% were determined in enantioconvergent reactions using lower temperatures. The EH activities of whole cells were found to be within the range of 74–125% of the wild-type activity for all investigated variants. We show in this report that the metagenome-derived Kau2 EH is amenable to the redesign of its enantioselectivity and regioselectivity properties by directed evolution using a homology model as a guide. The generated enzyme variants should be useful for the production of the chiral building blocks (R)-para-Chlorostyrene oxide and (R)-para-Chlorophenylethane-1,2-diol.     

Pooling for improved screening of combinatorial libraries for directed evolution

Following diversity generation in combinatorial protein engineering, a significant amount of effort is expended in screening the library for improved variants. Pooling, or combining multiple cells into the same assay well when screening, is a means to increase throughput and screen a larger portion of the library with less time and effort. We have developed and validated a Monte Carlo simulation model of pooling and used it to screen a library of beta-galactosidase mutants randomized in the active site to increase their activity toward fucosides. Here, we show that our model can successfully predict the number of highly improved mutants obtained via pooling and that pooling does increase the number of good mutants obtained. In unpooled conditions, we found a total of three mutants with higher activity toward p-nitrophenyl-beta-D-fucoside than that of the wild-type beta-galactosidase, whereas when pooling 10 cells per well we found a total of approximately 10 improved mutants. In addition, the number of "supermutants", those with the highest activity increase, was also higher when pooling was used. Pooling is a useful tool for increasing the efficiency of screening combinatorial protein engineering libraries.     

Key residues for controlling enantioselectivity of Halohydrin dehalogenase from Arthrobacter sp. strain AD2, revealed by structure-guided directed evolution

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and β-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E(S)] = 1.7 to R enantioselectivity [E(R)] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E(S) > 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.     

Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution

This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38 U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55 °C and 50 °C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60 °C and apparent melting temperature of reBaxA50 were 9.74 min and 89.15 °C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.     

Fluorescent intensity of a novel NADPH-binding protein of Vibrio vulnificus can be improved by directed evolution

Blue fluorescent protein, BfgV, found from Vibrio vulnificus CKM-1, fluoresces through augmenting the intrinsic fluorescence of bound NADPH. Random mutagenesis and DNA shuffling were applied to increase the fluorescent intensity of BfgV. The wild type bfgV gene was subjected to four cycles of mutagenesis processes. A prominent D7 mutant protein had fluorescent intensity four times larger than wild type BfgV. The emission wavelength of this mutant protein appeared at 440 nm, which was 16 nm shorter than that of BfgV. There were eight amino acid substitutions in D7. As these substitutions were assigned to the modeled 3D structure of BfgV, three of them, V83M, G176S, and E179K, were shown to be located around NADPH-binding site. Time course analysis indicated the synthesis of D7 protein and fluorescent expression in Escherichia coli transformants were synchronic. This property was different from that of wild type GFP.     

Enantioselective biocatalysis optimized by directed evolution

Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.     

Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity

Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli, which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p-nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.     

Analysis of novel soluble chromate and uranyl reductases and generation of an improved enzyme by directed evolution

Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, (Tyr)128(Asn), was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased.     

Optimizing the search algorithm for protein engineering by directed evolution

An in silico protein model based on the Kauffman NK-landscape, where N is the number of variable positions in a protein and K is the degree of coupling between variable positions, was used to compare alternative search strategies for directed evolution. A simple genetic algorithm (GA) was used to model the performance of a standard DNA shuffling protocol. The search effectiveness of the GA was compared to that of a statistical approach called the protein sequence activity relationship (ProSAR) algorithm, which consists of two steps: model building and library design. A number of parameters were investigated and found to be important for the comparison, including the value of K, the screening size, the system noise and the number of replicates. The statistical model was found to accurately predict the measured activities for small values of the coupling between amino acids, K 相似文献   

Fluorescent assay for directed evolution of perhydrolases

Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the μM range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4'-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k(cat) for perhydrolytic activity compared with wild type.     

Improved solubility of TEV protease by directed evolution

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.     

A directed molecular evolution approach to improved immunogenicity of the HIV-1 envelope glycoprotein

A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences.     

产海因酶的菌种筛选和产酶条件的研究

利用5-苄海因作为唯一氮源法筛选高产海因酶的菌种,从本实验室保存的221株菌种中筛选出12株具有不对称水解5-苄海因生成N-氨甲酰基-苯丙氨酸的菌株,其中假单胞菌（Pseudomonassp.)X4-49具有较高的产酶活力,对此菌的产酶条件的研究表明,产酶的最佳碳源为甘油,最佳氮源为蛋白胨,最佳诱导物为苄海因,尿嘧啶,苄海因作为诱导物的有效浓度为0.2%,产酶的最适培养基的初始pH为7.0。培养条件为33℃,13h。     

Diversifying carotenoid biosynthetic pathways by directed evolution.

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.