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At least 217 records · Page 12Linked to original sources
PubMed2006

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.

Amino Acid Sequence↗
PubMed2001

Enzyme engineering: rational redesign versus directed evolution.

Enzyme engineering is undergoing the most profound and exciting transformation in its history (1). It promises unprecedented expansion in the scope and applications of modified or improved enzymes with desired physical and catalytic properties. Two complementary strategies are currently available: rational redesign (2-5) and directed evolution (6,7). Although both approaches have met with great success, each has limitations. In this article, the perspectives for these enzyme-engineering strategies are discussed briefly.

Enzymes↗
PubMed2001

Changing the substrate specificity of cytochrome c peroxidase using directed evolution.

Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Using a combination of DNA shuffling and saturation mutagenesis, mutants were isolated which possessed more than 20-fold increased activity against ABTS and a 70-fold increased specificity toward ABTS compared to the natural substrate. In contrast, activities against another small organic molecule, guaiacol, were not significantly affected. Mutations at residues Asp224 and Asp217 were responsible for this increase in activity. These two residues are located on the surface of the protein and not in the direct vicinity of the distal cavity of the peroxidase, where small organic substrates are believed to be oxidized. Mutations at position Asp224 also lead to an increased amount of the active holoenzyme expressed in Escherichia coli, favoring the selection of these mutants in the employed colony screen. Possible explanations for the effect of the mutations on the in vitro activity of CCP as well as the increased amount of holoenzyme are discussed.

Base Sequence↗
PubMed1999

Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay.

In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).

Cell Division↗
PubMed2006

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling.

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.

Binding Sites↗
PubMed2004

Novel methods for directed evolution of enzymes: quality, not quantity.

In the past decade methods of directed molecular evolution have proven revolutionary in protein engineering. An increasing number of powerful new combinatorial techniques have joined rational design methods as effective tools for the manipulation and tailoring of biocatalysts. More and more, research in this maturing field is focusing on the quality and comprehensiveness of library construction and analysis. Additionally, in-depth studies have begun to highlight the underlying evolutionary mechanisms, limitations, and consequences of the various methodologies. Together, these investigations are creating a framework for future engineering projects.

Algorithms↗
PubMed2026

Directed evolution of Lactiplantibacillus plantarum for utilizing ethanol to produce postbiotics.

Alcohol is a recognized carcinogen worldwide. In this study, we aimed to utilize probiotics to metabolize ethanol and produce postbiotics. Initially, we identified a lactic acid bacteria community in kimchi with excellent probiotic activity. By employing our previously developed directed evolution techniques, a Lactiplantibacillus plantarum mutant with safe characteristics and an ethanol utilization capacity of 40 g/L and 0.15 g/L/OD was obtained. Genome sequencing and RT-qPCR analysis revealed the up-regulated expression of alcohol dehydrogenase and aldehyde dehydrogenase genes greatly contributed to ethanol utilization. Furthermore, the mutant strain demonstrated marked superiority in producing postbiotics, including antimicrobial peptides and beneficial organic acids such as lactic acid, phenyllactic acid, succinic acid, and indole-3-lactic acid. In the ethanol-fed fermentation process, the mutant strain achieved a lactic acid yield of 8.47 g/L and a carbon conversion rate of 21.8%. In vivo testing further validated its safety and ability to assist alcohol metabolism.

Adaptive laboratory evolution↗
PubMed2003

Creation of the first anomeric D/L-sugar kinase by means of directed evolution.

Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as d-galacturonic acid, d-talose, l-altrose, and l-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.

Carbohydrate Sequence↗
PubMed2005

Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles.

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

Amino Acid Sequence↗
PubMed1998

Directed evolution of an aspartate aminotransferase with new substrate specificities.

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.

Amino Acid Sequence↗
PubMed2002

Directed evolution of the site specificity of Cre recombinase.

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre. Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities. This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.

Amino Acid Sequence↗
PubMed2000

Directed evolution of beta -glucosidase A from Paenibacillus polymyxa to thermal resistance.

The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C. We have isolated random mutations that enhance the thermoresistance of the enzyme. Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a beta-glucosidase with a half-life of 12 min at 65 degrees C, in the range of resistance of thermophilic enzymes. No significant alteration of the kinetic parameters of the enzyme was observed. One of the mutants isolated in the screening for thermoresistant beta-glucosidase had the same resistance to denaturation as the wild type. This mutation caused the accumulation of enzyme in E. coli, probably due to its lower turnover. The structural changes responsible for the properties of the mutant enzymes have been analyzed. The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.

Adaptation, Physiological↗
PubMed2000

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.

Bacillus megaterium↗
PubMed2002

Directed evolution of a genetic circuit.

The construction of artificial networks of transcriptional control elements in living cells represents a new frontier for biological engineering. However, biological circuit engineers will have to confront their inability to predict the precise behavior of even the most simple synthetic networks, a serious shortcoming and challenge for the design and construction of more sophisticated genetic circuitry in the future. We propose a combined rational and evolutionary design strategy for constructing genetic regulatory circuits, an approach that allows the engineer to fine-tune the biochemical parameters of the networks experimentally in vivo. By applying directed evolution to genes comprising a simple genetic circuit, we demonstrate that a nonfunctional circuit containing improperly matched components can evolve rapidly into a functional one. In the process, we generated a library of genetic devices with a range of behaviors that can be used to construct more complex circuits.

DNA↗
PubMed1978

Regulation of newly evolved enzymes. IV. Directed evolution of the Ebg repressor.

In Escherichia coli, the wild-type repressor of ebg (evolved beta-galactosidase) enzyme synthesis, specified by the ebgR+ gene, responds very weakly to lactulose (fructose-beta-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 X 10(-10) . EBGR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

Biological Evolution↗
PubMed1981

Changes in the substrate specificities of an enzyme during directed evolution of new functions.

Wild-type ebg enzyme, the second beta-galactosidase of Escherichia coli K12, does not permit growth on lactose. As part of a study of the evolution of new enzymatic functions, I have selected, from a lacZ deletion strain, a variety of spontaneous mutants that grow on lactose and other beta-galactoside sugars. Single point mutations in the structural gene ebgA alter the enzyme so that it hydrolyzes lactose or lactulose effectively; two mutations in ebgA permit galactosylarabinose hydrolysis, while three mutations are required for lactobionic acid hydrolysis. Wild-type ebg enzyme and 16 functional mutant ebg enzymes were purified and analyzed kinetically to determine how the substrate specificities had changed during the directed evolution of these new functions. The specificities for the biologically selected substrates generally increased by at least an order of magnitude via increased Vmax and decreased Km for the substrate. These changes were very specific for the selected substrate, often being accompanied by decreased specificities for other related substrates. The single, double, or triple substitutions in the enzymes did not detectably alter the thermal stability of ebg enzyme.

Biological Evolution↗
PubMed1999

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling.

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.

Amino Acid Sequence↗