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PubMed2002

Does experimental evolution reflect patterns in natural populations? E. coli strains from long-term studies compared with wild isolates.

Our results show that experimental evolution mimics evolution in nature. In particular, only 1,000 generations of periodic recombination with immigrant genotypes is enough for linkage disequilibrium values in experimental populations to change from a maximum linkage value to a value similar to the one observed in wild strains of E. coli. Our analysis suggests an analogy between the recombination experiment and the evolutionary history of E. coli; the E. coli genome is a patchwork of genes laterally inserted in a common backbone, and the experimental E. coli chromosome is a patchwork where some sites are highly prone to recombination and others are very clonal. In addition, we propose a population model for wild E. coli where gene flow (recombination and migration) are an important source of genetic variation, and where certain hosts act as selective sieves; i.e., the host digestive system allows only certain strains to adhere and prosper as resident strains generating a particular microbiota in each host. Therefore we suggest that the strains from a wide range of wild hosts from different regions of the world may present an ecotypic structure where adaptation to the host may play an important role in the population structure.

Animals↗

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↗

PubMed2000

Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA.

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, Y. P., and Chen, K. Y. (1997) Biol. Signals 6, 166-174). Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) m. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in > 400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5'-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.

Base Sequence↗

PubMed2006

Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity.

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.

Amino Acid Sequence↗

PubMed2025

Adaptive laboratory evolution of Saccharomyces cerevisiae CEN.PK 113-7D to enhance ethanol tolerance.

Saccharomyces cerevisiae is a widely used yeast for industrial production of ethanol. However, elevated ethanol, temperature, and osmotic stress adversely affect fermentation efficiency. In this study, adaptive laboratory evolution for S. cerevisiae CEN.PK 113-7D on higher concentrations of ethanol was performed. After 144 days, the maximum specific growth rate (µmax) increased from 0.0240 to 0.1150 h-1 for the strain evolved on 9% v/v ethanol, and from 0.0002 to 0.0530 h-1 for the strain evolved on 11% v/v ethanol, and the specific glucose uptake rate increased by 30%. The strain evolved on 11% ethanol produced 94.5 g/L ethanol in a fermentation as compared to 78.5 g/L production by a non-evolved strain. By whole-genome sequencing of the evolved clones, we identified multiple coding mutations in genes involved in processes such as stress response, cell growth regulation, pentose phosphate pathway, lipid synthesis, and redox balance. The selected mutations in RKI1, CYC2, ANR2, RGA2, RGA1, LPX1, and LRE1 genes were validated by introducing them in the nonevolved yeast, showing 1.7-5-fold growth improvement at 9% ethanol (P < 0.05). Notably, RGA2, RGA1 and LPX 1 carried an identical missense mutation across three independent clones. The RKI1I208V mutant showed the highest ethanol tolerance, while CYC2N342A achieved the highest ethanol production.

Ethanol↗

PubMed2002

Directed evolution of barnase stability using proteolytic selection.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Amino Acid Sequence↗

PubMed2005

Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation.

We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.

Chryseobacterium↗

PubMed2004

Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.

Animals↗

PubMed2004

Covalent DNA display as a novel tool for directed evolution of proteins in vitro.

We present a novel method for the directed evolution of polypeptides, which combines in vitro compartmentalization and covalent DNA display. A library of linear DNA fragments is co-packaged with an in vitro transcription/translation mixture in the compartments of a water-in-oil emulsion. Experimental conditions are adjusted so that, in most cases, one compartment contains one DNA molecule. The DNA fragments encode fusion proteins containing a DNA-methyltransferase (M.Hae III), which can form a covalent bond with a 5-fluorodeoxycytidine base at the extremity of the DNA fragment. The resulting library of DNA-protein fusions is extracted from the emulsion and DNA molecules displaying a protein with desired binding properties are selected from the pool of DNA-protein fusions by affinity panning on target antigens. We applied this methodology in model selection experiments, using specific ligands for the capture of peptides and globular proteins bound to DNA. We observed enrichment factors >1000-fold for selections performed in separate emulsions and up to 150-fold for selections performed using mixtures of DNA molecules. M.Hae III could be fused to small globular proteins (such as calmodulin and fibronectin domains), which are ideally suited for the generation of combinatorial libraries and for the isolation of novel binding specificities.

DNA Adducts↗

PubMedDate not supplied

Estimation of the mutation rate during error-prone polymerase chain reaction.

Error-prone polymerase chain reaction (PCR) is widely used to introduce point mutations during in vitro evolution experiments. Accurate estimation of the mutation rate during error-prone PCR is important in studying the diversity of error-prone PCR product. Although many methods for estimating the mutation rate during PCR are available, all the existing methods depend on the assumption that the mutation rate is low and mutations occur at different places whenever they occur. The available methods may not be applicable to estimate the mutation rate during error-prone PCR. We develop a mathematical model for error-prone PCR and present methods to estimate the mutation rate during error-prone PCR without assuming low mutation rate. We also develop a computer program to simulate error-prone PCR. Using the program, we compare the newly developed methods with two other methods. We show that when the mutation rate is relatively low (< 10(-3) per base per PCR cycle), the newly developed methods give roughly the same results as previous methods. When the mutation rate is relatively high (> 5 x 10(-3) per base per PCR cycle, the mutation rate for most error-prone PCR experiments), the previous methods underestimate the mutation rate and the newly developed methods approximate the true mutation rate.

Biometry↗

PubMed2003

Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX.

We developed an integrated method to identify aptamers with only 10 fixed nucleotides through ligation and removal of primer binding sites within the systematic evolution of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach was validated by identifying a Spiegelmer ('mirror-image aptamer') that inhibits the action of the migraine-associated target calcitonin gene-related peptide 1 (alpha-CGRP) with an IC50 of 3 nM at 37 degrees C in cell culture. Aptamers are oligonucleotide ligands that can be generated to bind to targets with high affinity and specificity. Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic acid libraries that are composed of a central randomized region and additional fixed primer binding sites with approximately 30-40 nt. The identified sequences are usually not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization of aptamers and economical production. If the terminal primer binding sites are part of the target recognizing domain, truncation of aptamers has proven difficult and laborious. Tailored-SELEX results in short sequences that can be tested more rapidly in biological systems. Currently, our identified CGRP binding Spiegelmer serves as a lead compound for in vivo studies.

Alkalies↗

PubMed2005

Enhancement of the efficiency of secretion of heterologous lipase in Escherichia coli by directed evolution of the ABC transporter system.

The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

ATP-Binding Cassette Transporters↗

PubMed2001

Evolution of genetic variability in a population of the edible snail, Helix aspersa Müller, undergoing domestication and short-term selection.

The evolution of genetic variability is studied in six successive generations of a population originating from wild Helix aspersa. During the first three generations (G1 to G3), no artificial selection was applied. During the next three generations (G4 to G6), two lines were reared: a control line (C) and a line (S) selected for increased adult weight. Genetic variability is described by genealogical parameters (inbreeding, number of founders, effective number of founders and ancestors, effective number of remaining genomes) and by the additive genetic variance in adult weight. A large decrease in all parameters was observed between G1 and G2, suggesting strong natural selection: additive genetic variance in adult weight (transformed data) decreased from 0.0119 +/- 3.8 x 10(-3) to 0.0070 +/- 1.7 x 10(-3) (P < 0.05) and effective number of ancestors from 97.4 to 67.0. Selection also caused a large decrease during the first generation: additive genetic variance was 0.0079 +/- 2.1 x 10(-3) in G3 and 0.0040 +/- 1.1 x 10(-3) after the first selection cycle (P < 0.02). At the same time, the effective number of ancestors decreased from 59.2 to 29.5 and 24.2. This decrease is consistent with the theory of selection and the Bulmer effect.

Animals↗

PubMed2007

Versatile DNA fragmentation and directed evolution with nucleotide exchange and excision technology.

Mimicking natural evolution by DNA shuffling is a commonly used method for the optimization of DNA and protein properties. Here, we present an advancement of this approach whereby a gene library is amplified using a standard polymerase chain reaction (PCR), but incorporates dUTP as a fragmentation-defining exchange nucleotide, together with the four standard dNTPs. Incorporated uracil bases are excised using uracil-DNA-glycosylase, and the DNA backbone subsequently is cleaved with piperidine. This oligonucleotide pool is then reassembled with an internal primer extension procedure using a proofreading polymerase to increase yield, and, finally, is amplified by PCR. Denaturing polyacrylamide urea gels demonstrate this method to produce adjustable fragmentation size ranges dependent on the dUTP:dTTP ratios. Using the model protein, chloramphenicol acetyltransferase I, the sequencing of shuffled gene libraries based on a PCR containing 33% dUTP revealed a low mutation rate, of approx 0.1%, with an average parental fragments size of 86 bases, even without the use of a fragment-size separation. Nucleotide exchange and excision technology (NExT) DNA shuffling is, thus, reproducible and easily executed, making it superior to competing techniques. Additionally, NExT fragmentation outcome can be predicted using the computer software, NExTProg.

Chloramphenicol O-Acetyltransferase↗

PubMed1999

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.

Base Sequence↗

PubMed2003

Nitrogen-regulated hypermutator strain of Synechococcus sp. for use in in vivo artificial evolution.

Artificially evolved variants of proteins with roles in photosynthesis may be selected most conveniently by using a photosynthetic organism, such as a cyanobacterium, whose growth depends on the function of the target protein. However, the limited transformation efficiency of even the most transformable cyanobacteria wastes much of the diversity of mutant libraries of genes produced in vitro, impairing the coverage of sequence space. This highlights the advantages of an in vivo approach for generating diversity in the selection organism itself. We constructed two different hypermutator strains of Synechococcus sp. strain PCC 7942 by insertionally inactivating or nutritionally repressing the DNA mismatch repair gene, mutS. Inactivation of mutS greatly increases the mutation rate of the cyanobacterium's genes, leading to an up-to-300-fold increase in the frequency of resistance to the antibiotics rifampin and spectinomycin. In order to control the rate of mutation and to limit cellular damage resulting from prolonged hypermutation, we placed the uninterrupted mutS gene in the cyanobacterial chromosome under the transcriptional control of the cyanobacterial nirA promoter, which is repressed in the presence of NH(4)(+) as an N source and derepressed in its absence. By removing or adding this substrate, hypermutation was activated or repressed as required. As expected, hypermutation caused by repression in PnirA-mutS transformants led to an accumulation of spectinomycin resistance mutations during growth.

Adenosine Triphosphatases↗

PubMed2006

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus.

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.

Ascomycota↗

PubMed2004

Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire.

The Agrobacterium sp. beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.

Binding Sites↗