Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “directed evolution”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources
PubMed1996

One dimensional chemical shift modulated correlation spectroscopy of single-crystals.

A simple 1D modulation method is presented for determining the spatial correlation of chemical shifts in two different single-crystal orientations. This proposed chemical shift modulated correlation (CSMC) spectroscopy utilizes a Jeener pulse sequence to give 1D spectra containing peaks that are amplitude modulated by the chemical shifts associated with the single-crystal's evolution orientation. Conversely, the spectral frequencies in these 1D CSMC spectra designate the shifts in the crystal's orientation during the detection period. By observing the amplitude modulation of these spectral frequencies, the spatial correlation between the two chemical environments can be established for a specific nucleus from a single 1D spectrum. The CSMC procedure finishes by acquiring a normal 1D static spectrum for the crystal oriented in the evolution direction, thereby providing very accurate evolution frequencies approximated by the amplitude modulation in the CSMC spectra. The CSMC method improves the time efficiency over both the traditional goniometer method and the 2D chemical shift-chemical shift correlation method, and therefore is beneficial for the study of samples with longer T1 values.

Algorithms↗
PubMed2001

Protein-fold evolution in the test tube.

Currently, the combination of library selection and directed evolution is the most powerful approach for finding proteins with novel folds or functions. In the past, most studies concentrated either on protein scaffolds with a given fold or on short peptides. With the recent development of potent in vitro selection and evolution techniques, the screening of much larger sequence space is possible, allowing for the de novo generation of proteins.

Adenosine Triphosphate↗
PubMed1997

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Carboxylic Ester Hydrolases↗
PubMed2001

Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate.

We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only approximately 4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and approximately 20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.

Amino Acid Sequence↗
PubMed2026

Disruption of efflux activity reduces biofilm formation through multiple pathways.

Free-swimming bacteria must undergo large-scale changes in gene expression to form structured, aggregated biofilm communities. These regulatory changes are susceptible to environmental stimuli such as exposure to antimicrobials, which can affect adhesion, biofilm matrix production, pathogenicity and multidrug susceptibility. Previously, we found that genetic or chemical inactivation of efflux activity in Escherichia coli and Salmonella Typhimurium disrupts biofilm formation with a wide range of pathways sensitive to efflux inhibition, including reduced expression of csgD, a major regulator of biofilm matrix production. How the regulatory networks controlling efflux activity and biofilm formation overlap and how perturbing efflux impacts biofilm formation is still unclear. To address this, we used a combination of directed evolution experiments and large-scale functional genomics screens (TraDIS-Xpress) to identify the genes and pathways affecting efflux activity and biofilm formation in Salmonella enterica serovar Typhimurium and E. coli. This work describes the landscape of pathways linking efflux activity and biofilm formation. Whilst no singular gene or pathway was found to control the link between the two phenotypes, we propose changes in membrane potential following efflux inactivation are sensed through multiple response regulators that each in turn contribute to repression of biofilm development. These include the two-component signal transduction system EnvZ-OmpR and AraC/XylS family transcriptional regulators, RamA and MarA, which have extensive overlapping regulons and demonstrate high degrees of functional redundancy. This work deepens our understanding of the regulatory networks governing efflux activity and biofilm formation in Enterobacteriaceae and highlights the level of overlapping regulation and functional redundancy between them.

Salmonella typhimurium↗
PubMed2000

Information dynamics of in vitro selection-amplification systems.

Selection-amplifications systems provide a means of engineering biomacromolecules with new properties. The combination of stringent functional selection with the ability to amplify single molecules confers great specificity on the evolving population. Yet such systems like many complicated chemical kinetic mechanisms can show a range of unstable and metastable behavior. These instabilities can be investigated using the Shannon entropy of the evolving population. It is shown that the Shannon entropy provides a Lyapounov function for exploring dynamic stability. A simple model of in vitro evolution is presented and stability conditions are established. It is seen that fairly simple directed evolution models can exhibit a range of dynamical behavior.

Directed Molecular Evolution↗
PubMed1997

Isolation of novel ribozymes that ligate AMP-activated RNA substrates.

BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

Adenosine Monophosphate↗
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↗
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↗
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↗
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↗
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↗
PubMed2001

Stability, catalytic versatility and evolution of the (beta alpha)(8)-barrel fold.

The (beta alpha)(8)-barrel is a versatile single-domain protein fold that is adopted by a large number of enzymes. The (beta alpha)(8)-barrel fold has been used as a model to elucidate the structural basis of protein thermostability and in studies to interconvert catalytic activities or substrate specificities by rational design or directed evolution. Recently, the (beta alpha)(4)-half-barrel was identified as a possible structural subdomain.

Catalysis↗