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Results for “directed evolution”

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At least 541 records · Page 30Linked to original sources
PubMed2005

Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA.

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Bacterial Proteins↗
PubMed2003

Controlling chirality.

New strategies are continually being developed for using enzymes to efficiently catalyse the enantioselective synthesis of chiral non-racemic compounds. Alternatives to asymmetric synthesis or kinetic resolution include dynamic kinetic resolution, deracemisation and enantioconvergent transformations. Moreover, a much greater understanding is being developed of the parameters that can affect the stereochemical outcome of the reaction (e.g. solvent, substrate design, immobilization and directed evolution).

Catalysis↗
PubMed1999

Novel approaches for discovering industrial enzymes.

New technologies for enzyme discovery are changing the rules of the game for industrial biocatalysis. More kinds of enzymes are available, their hardiness is increasing, and their costs are coming down. These changes are the key drivers for a rebirth of interest in industrial applications of enzymes. The major enabling discovery approaches include screening of biodiversity, genomic sequencing, directed evolution and phage display.

Directed Molecular Evolution↗
PubMed1998

Combinatorial protein design by in vitro recombination.

DNA recombination is a powerful engine for the creation of new phenotypes. Recently, methods for in vitro DNA recombination (DNA shuffling) have been developed and applied to the evolution of novel molecules in the laboratory. An exciting new development is the shuffling of homologous genes to create diversity for directed evolution.

DNA↗
PubMed2004

Evolving haloalkane dehalogenases.

Mechanistic insight into the biochemistry of carbon-halogen bond cleavage is rapidly growing because of recent structural, biochemical and computational studies that have provided further insight into how haloalkane dehalogenases achieve their impressive catalytic activity. An occluded water-free active-site cavity together with strong hydrogen bond donating groups reduce the transition state energy barrier compared with that of the non-enzymatic reaction in water. Even though all known haloalkane dehalogenases belong to the alpha/beta-hydrolase fold family, there are interesting differences in mechanistic and kinetic details, as shown by properties of mutant enzymes and transient-state kinetic studies. To improve enzymatic degradation of some environmentally important recalcitrant compounds, site-directed mutagenesis and directed-evolution studies are being done.

Catalysis↗
PubMed2005

High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS). Earlier in vivo studies have demonstrated that Ebg, a protein of unknown function, can evolve to allow Escherichia coli lacking the lacZ beta-galactosidase gene to grow on lactose. Here we demonstrate that we can evolve Ebg into an enzyme with significant beta-galactosidase activity in vitro. Only two specific mutations were ever seen to provide this improvement in Ebg beta-galactosidase activity in vivo. In contrast, nearly all the improved beta-galactosidases selected in vitro resulted from different mutations.

Directed Molecular Evolution↗
PubMed2002

Microbial carboxyl esterases: classification, properties and application in biocatalysis.

Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds and are widely distributed in animals, plants and microorganisms. Beside lipases, a considerable number of microbial carboxyl esterases have also been discovered and overexpressed. This review summarizes their properties and classification. Special emphasis is given on their application in organic synthesis for the resolution of racemates and prostereogenic compounds. In addition, recent results for altering their properties by directed evolution are presented.

Bacteria↗
PubMed1999

Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.

Archaeal Proteins↗
PubMed2001

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.

Amino Acids↗
PubMed2002

Construction of heme enzymes: four approaches.

Construction of enzymes that catalyze either desired reactions or exhibit desired substrate specificity is one of the goals of enzymatic study. Rational design of enzymes is an important approach in this field. Another, extremely different, methodology from rational design, directed evolution, has been rapidly developed over the past two years.

Binding Sites↗
PubMed2003

Speciation in multidimensional evolutionary space.

Adaptive dynamics in two-dimensional phenotype space is investigated by computer simulation. The model assumes Lotka-Voltera-type competition and a stochastic mutation process. The carrying capacity has a single maximum in the origin of the strategy space and the competition coefficient decreases with strategy difference. Evolutionary branching, an asexual analog of adaptive speciation, is observed with suitable parameters. The branching at the singular point, which is a fixed point of the directional evolution, may occur into two or three, but not more, directions. Further branchings may occur after the initial separation. The probability of three-branching is studied as a function of several parameters. We conclude that the two-way branching is the predominant mode of adaptive speciation.

Adaptation, Physiological↗
PubMed2000

Designed evolution of enzymatic properties.

By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis. Recent advances include the evolution of a novel catalytic activity using the alpha/beta barrel scaffold, evolution of a cofactor-free monooxygenase, and the engineering of regulatable enzymes. New screening systems for enantioselectivity and protein solubility, and the continuing stream of new methods for creating enzyme libraries further extend evolution's reach.

Biotechnology↗
PubMed2001

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes.

BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space. RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme. CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.

Aminohydrolases↗
PubMed2005

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

Cucumber Mosaic Virus Satellite↗
PubMed2006

A high-throughput screening assay for hydroxynitrile lyase activity.

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.

Aldehyde-Lyases↗
PubMed2000

Evolution in the test tube as a means to create enantioselective enzymes for use in organic synthesis.

A novel method for the creation of mutant enzymes showing increased levels of enantioselectivity in a given synthetic organic reaction is reviewed. It makes use of directed evolution and is therefore independent of structural or mechanistic aspects. Accordingly, known molecular biological methods for random mutagenesis coupled with the proper expression system and high-throughput screening methods for enantioselectivity form the basis of this new approach. An example is the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2% ee is increased to > 90% ee in just a few rounds of mutagenesis.

Directed Molecular Evolution↗
PubMed2006

Evolving sensitivity.

Engineering gene circuits with novel functions holds promise for broad applications in biology, engineering, and medicine. Directed evolution complements rational design as an important strategy for optimizing gene circuits and circuit elements.

Directed Molecular Evolution↗
PubMed2000

Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution.

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.

Adaptation, Physiological↗