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At least 343 records · Page 19Linked to original sources
PubMed2004

An evolutionary route to xylanase process fitness.

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).

Amino Acid Substitution↗
PubMed2001

Identification by saturation mutagenesis of a single residue involved in the alpha-galactosidase AgaB regioselectivity.

The alpha-galactosidase AgaB of Bacillus stearothermophilus displays a major 1,6 and a minor 1,3 regioselectivity. The wild-type enzyme was subjected to directed evolution (random mutagenesis and in vitro recombination) using a double screening strategy based on the elimination of the 1,6 regioselectivity and the analysis by TLC of the transglycosylation products. One of the AgaB mutants (E500) exhibited a new 1,2 regioselectivity and a rather high level of transglycosylation. The corresponding gene contains 10 mutations compared to the agaB gene and we demonstrated by saturation mutagenesis that the G442R substitution strongly contributes to the emergence of this new regioselectivity. Moreover, other single point mutations at this position led to new mutants displaying other kinds of regioselectivity demonstrating the importance of this position in the subtle kinetic control of transglycosylation.

Cloning, Molecular↗
PubMed2001

In-vitro selection of highly stabilized protein variants with optimized surface.

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.

Amino Acid Sequence↗
PubMed2000

Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases.

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.

Directed Molecular Evolution↗
PubMedDate not supplied

Fine affinity discrimination by yeast surface display and flow cytometry.

Yeast surface display is a eucaryotic system for the directed evolution of protein binding and stability. For antibody affinity maturation, achievable single-pass enrichment factors are a critical variable. Both reliable recovery of rare clones (yield) and effective differentiation between clones of only slightly improved affinity (purity) are paramount. To validate yeast display's purification potential, trial sorting experiments were performed. The D1.3 (anti-hen egg lysozyme) single chain variable fragment antibody and a 2-fold higher affinity mutant (M3) were each displayed on the surface of Saccharomyces cerevisiae. M3-displaying cells were mixed into the D1.3-displaying cells at a ratio of 1:1000. Cells were fluorescently labeled according to antigen equilibrium binding and then sorted using a flow cytometer. Single-pass enrichment of M3-displaying cells was 125-fold (+/- 65-fold). This level of performance is achievable because of the precision and reproducibility of optimal labeling conditions. This work further demonstrates the capability of yeast display for very fine discrimination between mutant clones of similar affinity. Because large improvements in affinity typically result from combinations of small changes, this capability to identify subtle improvements is essential for rapid affinity maturation.

Animals↗
PubMedDate not supplied

Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant).

Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p < 0.10) between KO11 and LY01 under each of six different growth conditions (p < 0.000001). Statistical evaluation of differentially expressed genes according to various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from choline and choline uptake, respectively). Addition of glycine (10 mM) increased ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11. Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of the cell envelope components were also noted, which may contribute to increased ethanol tolerance.

Directed Molecular Evolution↗
PubMedDate not supplied

[Ecosystem evolution: main stages and potential mechanisms].

Major differences between the Western and "Russian" (Zavarzin, 1995) paradigms in ecology and evolutionary biology are described. The "Russian" paradigm suggests that there exist two, rather than one, quite independent lineages--species evolution and ecosystem evolution. This is based on the idea that life may exist just as a nutrient cycle. The main terms and concepts of the "Russian" paradigm are defined more exactly. An attempt is made to develop this paradigm so that it would be possible to describe not only phenomenology, but also mechanisms of ecosystem evolution. To simplify evolutionary phenomena logically, it is suggested to use the concept of conditionally complete causal explanation (Lekevicius, 1984; 1985), i.e. deduce evolutionary mechanisms from major principles of functioning. This methodology is adapted to model the main stages of the evolution of nutrient cycles (3.8-2.0 bln. y.a.) and the appearance and evolution of biophagy (1.7-0 bln. y.a.). Based on a multitude of examples, it is shown that these are functional constraints that are the forces directing evolution; those constraints emerge during the interaction of organisms and while the latter interact with the abiotic environment. Since the structure of an ecosystem is non-rigid, each species is able to accumulate features useful to both an ecosystem and itself. Those are individuals that die and reproduce, whereas all structures, from macromolecules to ecosystems, evolve.

Animals↗
PubMed1999

Dynamic diversity and small-molecule evolution: a new paradigm for ligand identification.

A longstanding goal of organic, medicinal and bioorganic chemists has been the discovery of efficient methods for designing or identifying biologically active compounds. Recently, several groups have reported using the directed evolution of combinatorial libraries as a new method of identifying compounds capable of binding tightly to a target molecule. Although significant development remains to be done, the initial results suggest that dynamic diversity and associated selection methods will prove to be valuable additions to the drug-discovery process.

Biological Evolution↗
PubMed2000

Directionality theory and the evolution of body size.

Directionality theory, a dynamic theory of evolution that integrates population genetics with demography, is based on the concept of evolutionary entropy, a measure of the variability in the age of reproducing individuals in a population. The main tenets of the theory are three principles relating the response to the ecological constraints a population experiences, with trends in entropy as the population evolves under mutation and natural selection. (i) Stationary size or fluctuations around a stationary size (bounded growth): a unidirectional increase in entropy; (ii) prolonged episodes of exponential growth (unbounded growth), large population size: a unidirectional decrease in entropy; and (iii) prolonged episodes of exponential growth (unbounded growth), small population size: random, non-directional change in entropy. We invoke these principles, together with an allometric relationship between entropy, and the morphometric variable body size, to provide evolutionary explanations of three empirical patterns pertaining to trends in body size, namely (i) Cope's rule, the tendency towards size increase within phyletic lineages; (ii) the island rule, which pertains to changes in body size that occur as species migrate from mainland populations to colonize island habitats; and (iii) Bergmann's rule, the tendency towards size increase with increasing latitude. The observation that these ecotypic patterns can be explained in terms of the directionality principles for entropy underscores the significance of evolutionary entropy as a unifying concept in forging a link between micro-evolution, the dynamics of gene frequency change, and macro-evolution, dynamic changes in morphometric variables.

Animals↗
PubMed2002

Transition rates between specialization and generalization in phytophagous insects.

Although most species of animals exhibit specialized patterns of resource use, it is unclear whether specialization evolves at a faster rate than generalization. To test this hypothesis, transition rates toward specialization and toward generalization were estimated using phylogenies from 15 groups of phytophagous insects. Among the groups studied, maximum-likelihood analyses showed that the forward transition rate from generalization to specialization was significantly higher than the reverse transition rate from specialization to generalization (mean ratio of forward to reverse transition rate = 1.47 using uniform branch lengths and 1.76 using Grafen branch lengths). Although phylogenetic conservatism of host-plant use is common, the results suggest that the evolution of specialization is a highly dynamic process. For example, higher transitions rates both toward and away from specialization as well as equal transition rates were inferred. Collectively, the results reveal a tendency for directional evolution toward increased specialization but also indicate that specialization does not always represent an evolutionary dead-end that strongly limits further evolution.

Animals↗
PubMed2001

Rapid evolution of novel traits in microorganisms.

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

Dimethylformamide↗
PubMed2003

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily.

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.

Amino Acid Sequence↗
PubMed2003

Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein.

We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130-150 amino acids named Shadoo (Japanese shadow), predicted to be extracellular and GPI-anchored. The SPRN gene was found in fish (zebrafish, Fugu) and mammals (mouse, rat, human). Conservation of order and transcription orientation of two proximal genes between fishes and mammals strongly indicates gene orthology. Sequence comparison shows: a highly conserved N-terminal signal sequence; Arg-rich basic region containing up to six tetrarepeats of consensus XXRG (where X is G, A or S); a hydrophobic region of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing a conserved glycosylation motif; and a C-terminal peptide predicted to be a signal sequence for glycophosphotidylinositol (GPI)-anchor attachment. Fish Shadoos (Sho) show well conserved sequences (identity 54%) over 106 amino acids (zebrafish length), and conservation among the mammalian sequences is very high (identity 81-96%). The fish and mammalian sequences are also well conserved, particularly for zebrafish, to beyond the end of the hydrophobic sequence (identity 41-53%, 78 amino acids, zebrafish length). The overall structure appears closely related to prion proteins (PrPs), although the C-terminal domains of Shos are quite different from those of PrPs, for which conformational changes in mammals are implicated in disease. The structural similarity is particularly interesting given recent reports of three new genes with similarities to PrPs found in Fugu (PrP-like, PrP-461/stPrP-1 and stPrP-2) and other fish, but for which direct evolution to higher vertebrate PrPs is unlikely and for which no other mammalian homologues have been found. Database information indicates expression of SPRN in embryo, brain and retina of mouse and rat, hippocampus of human, and in embryo and retina of zebrafish, and we directly confirmed a strikingly specific expression of the mammalian (human, mouse, rat) transcripts in whole brain. This result together with some common structural features led to the suggestive hypothesis of a possible functional link between mammalian PrP and Sho proteins.

Amino Acid Sequence↗
PubMed2002

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology.

An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.

DNA Primers↗
PubMed2003

How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001 or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.

Amino Acid Sequence↗
PubMed2003

Chemical complementation: small-molecule-based genetic selection in yeast.

Protein and metabolic engineering would greatly benefit from a general system linking the presence of a small molecule to the power of genetic selection. We use nuclear receptors to link the survival of Saccharomyces cerevisiae to the presence of small molecules through genetic selection, extending classical genetic complementation to a new "chemical complementation." In this system the Gal4 DNA-binding domain is fused to ligand-binding domains from two nuclear receptors, expressed in the strain PJ69-4A, and grown on plates containing known ligands for the receptors. Yeast survive on selective plates only in the presence of a nuclear receptor and the corresponding ligand. Mutagenesis can increase the sensitivity of chemical complementation. This system may be extended to engineer nuclear receptors for practically any small molecule through directed evolution coupled to genetic selection, and for performing metabolic engineering in yeast.

Antineoplastic Agents↗
PubMed1996

Particulate versus integrated evolution of the upper body in late pleistocene humans: a test of two models.

Evolutionary biologists are largely polarized in their approaches to integrating microevolutionary and macroevolutionary processes. Neo-Darwinians typically seek to identify population-level selective and genetic processes that culminate in macroevolutionary events. Epigeneticists and structuralists, on the other hand, emphasize developmental constraints on the action of natural selection, and highlight the role of epigenetic shifts in producing evolutionary change in morphology. Accordingly, the ways in which these paradigms view and address morphological contrasts between classes of related organisms differ. These paradigms, although seldomly explicitly stated, emerge in paleoanthropology as well. Considerations of postcranial morphological contrasts between archaic and modern humans typically fall into one of two broad interpretive models. The first derives from the neo-Darwinian perspective and holds that evolution in the postcranial skeleton was largely mosaic (operating in a particulate manner), and that temporal change in specific traits informs us about behavioral shifts or genetic evolution affecting isolated anatomical regions (i.e., adaptive behavioral inferences can be made from comparative studies of individual trait complexes). The alternative model follows from the epigeneticist paradigm and sees change in specific postcranial traits as correlated responses to change in overall body form (involving shifts in regulation of skeletal growth, or selective and developmental responses to broad adaptive shifts). By this view, integration of functional systems both constrains and directs evolution of various traits, and morphological contrasts inform us about overall change in body form related to change in such things as overall growth patterns, climatic adaptation, and technological dependency. These models were tested by confirmatory factor analysis using measures of upper body form and upper limb morphological traits in Eurasian Neandertal and early modern fossils and recent human samples. Results indicate (1) a model of morphological integration fits the data better than a model of no integration, but (2) this integration accounts for less than half of the variance in upper limb traits, suggesting a high degree of tolerance for particulate evolution in the context of an integrated upper body plan. Significant relationships were detected between joint shapes and body size, between humeral shaft shape and body size and chest shape, and between measures of biomechanical efficiency and robusticity. The observed morphological differences between late archaic and early modern humans reflect particulate evolution in the context of constraints imposed by genetic and morphological integration. While particulate approaches to interpreting the fossil record appear to be justified, attention must also be paid to delineating the nature and extent of morphological integration and its role in both constraining and producing observed patterns of variation between groups. Confirmatory factor analysis provides a means of examining trait covariance matrices, and serves as a useful method of identifying patterns of integration in morphology.

Biological Evolution↗
PubMed1998

Molecular evolution by staggered extension process (StEP) in vitro recombination.

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.

Bacillus subtilis↗