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At least 19 recordsLinked to original sources

PubMed2001

Improving the quality of industrially important enzymes by directed evolution.

Directed evolution is a new process for developing industrially viable biocatalysts. This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering. It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties. Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments. This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability.

Animals↗

PubMed2004

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.

Algorithms↗

PubMed2004

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution.

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.

Aeropyrum↗

PubMed2006

[Rational evolutionary design and modification of gene: a short-path of direct evolution].

Directed evolution is a powerful means in academic study and industrial application to modified and functionally improved proteins. There have been significant and impressive advances using the artificial method. Rational Evolutionary Design utilizes structural and sequence alignment information to create new genes and proteins. Rational Evolutionary Design has recently emerged as an attractive approach for studying function of proteins. The review mainly introduced four aspects: preferred codon usage, structural and sequence alignment, key domain analysis, site-directed recombination.

Directed Molecular Evolution↗

PubMed2003

Optimization of protein therapeutics by directed evolution.

Directed evolution is a broadly applicable technology platform that is ideally suited to address the need for protein optimization and to fully exploit the therapeutic potential of biologics. The approach takes advantage of the remarkable structural and functional plasticity of proteins and permits the rapid remodeling of biologics into new entities with improved functions. The ability to ameliorate virtually any characteristic of a protein can translate into significant clinical benefits, including decreased immunogenicity, higher potency, greater efficacy and improved safety profile, and can considerably increase the probability of successfully developing and commercializing biotherapeutics.

Animals↗

PubMed2005

Converting a {beta}-glycosidase into a {beta}-transglycosidase by directed evolution.

Directed evolution was applied to the beta-glycosidase of Thermus thermophilus in order to increase its ability to synthesize oligosaccharide by transglycosylation. Wild-type enzyme was able to transfer the glycosyl residue with a yield of 50% by self-condensation and of about 8% by transglycosylation on disaccharides without nitrophenyl at their reducing end. By using a simple screening procedure, we could produce mutant enzymes possessing a high transferase activity. In one step of random mutagenesis and in vitro recombination, the hydrolysis of substrates and of transglycosylation products was considerably reduced. For certain mutants, synthesis by self-condensation of nitrophenyl glycosides became nearly quantitative, whereas synthesis by transglycosylation on maltose and on cellobiose could reach 60 and 75%, respectively. Because the most efficient mutations, F401S and N282T, were located just in front of the subsite (-1), molecular modeling techniques were used to explain their effects on the synthesis reaction; we can suggest that repositioning of the glycone in the (-1) subsite together with a better fit of the acceptor in the (+1) subsite might favor the attack of a glycosyl acceptor in the mutant at the expense of water. Thus these new transglycosidases constitute an interesting alternative for the synthesis of oligosaccharides by using stable and accessible donor substrates.

Cellobiose↗

PubMed2004

Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution.

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.

Base Sequence↗

PubMed2000

Modeling DNA mutation and recombination for directed evolution experiments.

Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data.

Animals↗

PubMed2002

Generating segmental mutations in haloalkane dehalogenase: a novel part in the directed evolution toolbox.

Directed evolution techniques allow us to genuinely mimic molecular evolution in vitro. To enhance this imitation of natural evolutionary processes on a laboratory scale in even more detail, we developed an in vitro method for the generation of random deletions and repeats. The pairwise fusion of two fragments of the same gene that are truncated by exonuclease BAL-31 either at the 3' or 5' side results in a deletion or a repeat at the fusion point. Although in principle the method randomly covers the whole gene, it can also be limited to a predefined area in the sequence by controlling the level of the initial truncation. To test the procedure and to illustrate its potential, we used haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) as a model enzyme, since the adaptation of this enzyme towards new substrates is known to occur via the generation of this type of mutation. The results show that the mutagenesis method presented here is an effective tool for accessing formerly unexplorable sequence space and can contribute to the success of future directed evolution experiments.

Amino Acid Sequence↗

PubMed2006

Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution.

We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xyl(st), was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xyl(st) variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 degrees C. At 60 degrees C the wild-type enzyme was inactivated within 5 min, but Xyl(st) retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 degrees C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xyl(st) enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xyl(st). This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xyl(st). However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.

Bacillus subtilis↗

PubMed2001

Predicting the emergence of antibiotic resistance by directed evolution and structural analysis.

Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.

Amino Acid Sequence↗

PubMed1999

Directed evolution of biocatalysts.

Directed evolution is being used increasingly in academic and industrial laboratories to modify and improve important biocatalysts. Significant advances during this period of review include compartmentalization of genes and the in vitro translation apparatus in emulsions, as well as several impressive demonstrations of catalyst improvement. Shuffling of homologous genes offers a new way to utilize natural diversity in the evolution of novel catalysts.

Catalysis↗

PubMed2002

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.

Amino Acid Substitution↗

PubMed2001

Computationally focusing the directed evolution of proteins.

Directed evolution has proven to be a successful strategy for the modification of enzyme properties. To date, the preferred experimental procedure has been to apply mutations or crossovers randomly throughout the gene. With the emergence of powerful computational methods, it has become possible to develop focused combinatorial searches, guided by computer algorithms. Here, we describe several computational methods that have emerged to aid the optimization of mutant libraries, the targeting of specific residues for mutagenesis, and the design of recombination experiments.

Algorithms↗

PubMed2001

Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution.

With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.

Directed Molecular Evolution↗

PubMed2005

Directed evolution: selecting today's biocatalysts.

Directed evolution has become a full-grown tool in molecular biology nowadays. The methods that are involved in creating a mutant library are extensive and can be divided into several categories according to their basic ideas. Furthermore, both screening and selection can be used to target the enzyme towards the desired direction. Nowadays, this technique is broadly used in two major applications: (industrial) biocatalysis and research. In the first field enzymes are engineered in order to produce suitable biocatalysts with high catalytic activity and stability in an industrial environment. In the latter area methods are established to quickly engineer new enzymes for every possible catalytic step, thereby creating a universal biotechnological toolbox. Furthermore, directed evolution can be used to try to understand the natural evolutionary processes. This review deals with new mutagenesis and recombination strategies published recently. A full overview of new methods for creating more specialised mutant libraries is given. The importance of selection in directed evolution strategies is being exemplified by some current successes including the beta-lactam acylases.

Amidohydrolases↗

PubMed2003

Directional evolution for microsatellite size in maize.

Directional evolution in microsatellites is the tendency for microsatellites either to increase or to decrease in size over time between populations. We analyzed 99 microsatellite loci in a sample of 193 maize plants representing the entire pre-Columbian range of this crop for evidence of directional evolution. We took advantage of the known phylogeographic history of maize with the independent movement of maize from its ancestral location in Mexico to North and South America. We show that there is an increase in the average allele size of microsatellites in the geographically derived North and South American groups relative to the ancestral Mexican group. We also show that there is a negative correlation between average allele size and altitude in all three groups. Directional evolution in maize microsatellites can be explained by changes in the mutation rate over time and space, changes in the degree of mutational bias to a larger allele, demographic differences between the ancestral and geographically derived populations, and/or scenarios involving selection on microsatellite size. The occurrence of directional evolution for microsatellite size indicates that the estimation of population parameters with microsatellite data in maize should be done with caution.

Alleles↗

PubMed2006

Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.

CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.

Benzene Derivatives↗