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PubMed1997

Mode of mating preference and the direction of evolution in Drosophila.

Sexual (or ethological) isolation constitutes the most important class among different means of reproductive isolation in animal species. The phenomenon of sexual isolation has been extensively studied in the genus Drosophila. The degree of sexual isolation and the mode of mating preference are often used to indicate the phylogenetic relationship between the species and also their evolutionary sequence. Asymmetrical or one-sided mating preference observed in a number of Drosophila species groups has been used to elucidate the direction of evolution. Based on the mode of mating preference, different models which are even opposite to each other have been proposed by different investigators to predict the direction of evolution among closely related species. One-sided mating preference observed among the geographical strains of the same species has also been used to predict the direction of evolution. This review documents several cases of asymmetrical mating preference (both inter- and intraspecific) including the interesting cases reported by Indian workers and in the light of these observations of asymmetry in mating preference, the general applicability of different models is discussed.

Animals↗
PubMed2003

Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate.

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.

Binding Sites↗
PubMed2002

Directed molecular evolution: bridging the gap between genomics leads and commercial products.

Agricultural crops, engineered to express transgenic traits, have been rapidly adopted by farmers since the initial commercialization of this technology in 1996. However, despite nearly 20 years of research in agricultural biotechnology, only two product categories have achieved commercial success: plants containing transgenes conferring tolerance to herbicides and plants containing insecticidal protein genes derived from Bacillus thuringensis. A number of transgenic concepts, while exhibiting promising phenotypes in laboratory experiments, have failed to generate commercially viable crops. Many of the leads produced by modern integrative approaches to understanding plant biology will need further optimization to deliver economically viable crops. Directed molecular evolution represents a powerful technology to optimize newly discovered leads towards product objectives. In this review, we show by example how directed molecular evolution can be used to develop enabling technologies for plant biologists; how genes can be optimized to generate improved input traits such as those conferring insect tolerance, disease control and herbicide tolerance; and how plant quality can be altered to improve yield, produce novel industrial feedstocks and improve nutritional qualities.

Agriculture↗
PubMed2006

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

Allergens↗
PubMed2004

Directed enzyme evolution and selections for catalysis based on product formation.

Enzyme engineering by molecular modelling and site-directed mutagenesis can be remarkably efficient. Directed enzyme evolution appears as a more general strategy for the isolation of catalysts as it can be applied to most chemical reactions in aqueous solutions. Selections, as opposed to screening, allow the simultaneous analysis of protein properties for sets of up to about 10(14) different proteins. These approaches for the parallel processing of molecular information 'Is the protein a catalyst?' are reviewed here in the case of selections based on the formation of a specific reaction product. Several questions are addressed about in vivo and in vitro selections for catalysis reported in the literature. Can the selection system be extended to other types of enzymes? Does the selection control regio- and stereo-selectivity? Does the selection allow the isolation of enzymes with an efficient turnover? How should substrates be substituted or mimicked for the design of efficient selections while minimising the number of chemical synthesis steps? Engineering sections provide also some clues to design selections or to circumvent selection biases. A special emphasis is put on the comparison of in vivo and in vitro selections for catalysis.

Biotechnology↗
PubMed2005

The use of scFv-displaying yeast in mammalian cell surface selections.

Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.

Animals↗
PubMed2003

Teaching TetR to recognize a new inducer.

Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.

Amino Acid Sequence↗
PubMed2006

Molecular mechanisms of improvement of hydrolytic antibody 6D9 by site-directed mutagenesis.

We performed a series of site-directed mutagenesis experiments of catalytic antibody, 6D9, which hydrolyzes a prodrug of chloramphenicol, based on our previous directed evolution study [Takahashi et al. (2001) Nat. Biotechnol. 19, 563-567]. Since we previously found that the variants with a mutation of Ser(L27e)Tyr afforded a one order of magnitude increase in catalytic rate, we created a site-directed mutant containing this mutation. The resulting mutant, 6D9-Ser(L27e)Tyr, had 6.5-fold higher k(cat)/k(uncat) and 9.8-fold higher k(cat)/K(m) than wild-type 6D9. We also created 6D9-Thr(L27a)Pro, since this mutation occurred frequently in the previous directed evolution, and it had 2.1-fold higher k(cat)/k(uncat) and k(cat)/K(m) than 6D9. Kinetic and computational analyses suggest that Tyr at L27e contributes to transition-state stabilization, while Pro at L27a does not interact with the transition-state structure directly, but obviously contributes to enhanced catalytic activity. Including double mutants that combined favourable substitutions, we created seven site-directed mutants. However, none of them had higher catalytic activities than some of highly improved variants obtained in the previous directed evolution. The present study gives direct evidence that not only a specific amino acid residue which obviously contributes to transition-state stabilization, but also a group of amino acid residues working in concert is important for efficient catalysis of a given transformation.

Animals↗
PubMed2005

Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters.

Genetic vaccinations, gene therapy, and manufacturing of therapeutic proteins would benefit from promoter sequences that provide improved or prolonged expression levels. The cytomegalovirus (CMV) promoter is one of the most potent promoters known to date, and no previous examples of improved activity of this promoter by sequence mutagenesis have been reported. This study describes directed molecular evolution of CMV promoters derived from two human and two nonhuman primate strains of CMV by DNA shuffling and screening. Libraries of chimeric promoters were screened and analyzed for expression levels and immune responses, using plasmid DNA vectors encoding luciferase and beta-galactosidase. The results indicate that high functional diversity among CMV promoters can be generated, and the chimeric promoters selected after two rounds of DNA shuffling and particularly designed screening assays provided approximately 2-fold increased luciferase reporter gene expression and anti-beta-galactoside antibody response in vivo when compared with wild-type promoters. Sequence analysis of the shuffled promoters identified several mutations potentially contributing to the observed enhanced or reduced promoter activities and identified a 42-nucleotide region that appears obsolete for the functioning of the CMV promoter. Taken together, these data demonstrate the feasibility of generating diverse promoter sequences by DNA shuffling and screening methods, and provide novel structure- function information about CMV promoters. DNA shuffling and screening technologies provide a new approach to promoter optimization and development of optimal expression vectors for genetic vaccinations, gene therapy, and protein expression.

Animals↗
PubMed2001

Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case.

Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced-in the same region and based on the crystal structure of A. niger phytase-entire secondary structure elements. The replacement of one alpha-helix on the surface of A. terreus phytase by the corresponding stretch of A. niger phytase resulted in an enzyme with improved thermostability and unaltered enzymatic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein contained only a 31 amino acid stretch of the more stable parent enzyme. This report provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propose this method as an alternative to directed enzyme evolution if due to expression constraints the screening of large mutant populations is not feasible.

Amino Acid Sequence↗
PubMed2001

Mating asymmetry and the direction of evolution in the Hawaiian cricket genus Laupala.

Based on studies from native Hawaiian Drosophila, a model was proposed to explain sexual isolation and mating asymmetry, from which one could potentially infer the 'direction of evolution'. We examined sexual isolation between allopatric cricket species of the genus Laupala, another endemic Hawaiian insect with an elaborate mating system, to begin to explore the nature of sexual isolation and mating asymmetry in closely related Hawaiian organisms. We studied sexual isolation and mating asymmetry in two contrasts. First, an inter-island comparison, including L. makaio from the older island of Maui and L. paranigra from the younger island of Hawaii, and second, an intra-island (Hawaii) comparison, including L. nigra from the older volcano of Mauna Kea and L. paranigra with a primary distribution on the younger volcanoes of Mauna Loa and Kilauea. We used a 'no-choice' experimental design, pairing individual males and females in homospecific or heterospecific combinations. Several behavioural aspects of courtship (proportion of male singing, latency to male singing, production of spermatophores and courtship initiation speed) were quantified as well as the success or failure of matings. We demonstrate asymmetry in sexual isolation between reciprocal combinations of L. makaio and L. paranigra. This result is examined in light of the differences in courtship behaviour manifest in the experiments with these two species. We did not find evidence of asymmetry in sexual isolation between L. nigra and L. paranigra, although differences in courtship initiation speed were evident between reciprocal combinations of these two species. In addition to the geological argument that species on older islands and older volcanoes give rise to species on younger islands and younger volcanoes, we discuss phylogenetic evidence consistent with these biogeographic hypotheses of relationships among the focal taxa. The patterns of asymmetrical sexual isolation and mating asymmetry are consistent with those found in the native Hawaiian Drosophila.

Animals↗
PubMedDate not supplied

The effect of development on the direction of evolution: toward a twenty-first century consensus.

One of the most important questions in evolutionary biology is: what orients the evolutionary process? That is, what causes evolution to proceed toward certain developmental trajectories, and hence phenotypes, rather than others? In particular, there has been prolonged controversy over whether the direction of evolution is determined solely by external factors or whether the nature of the ontogenetic process, and the ways in which it can be altered by mutations in developmental genes, may also play a major role. Here, I examine this issue, concentrating on the following: the possible evolutionary orienting role of "developmental bias;" the question of whether selection can and/or will break bias; the extent to which bias is already incorporated in quantitative genetic studies; and ways of approaching the possible role of bias in the origin of evolutionary novelties. Finally, I suggest that developmental bias may provide a focal point for the coming together of conceptual and practical approaches to evo-devo.

Biological Evolution↗
PubMed2004

Detection of biological threats. A challenge for directed molecular evolution.

The probe technique originated from early attempts of Anton van Leeuwenhoek to contrast microorganisms under the microscope using plant juices, successful staining of tubercle bacilli with synthetic dyes by Paul Ehrlich and discovery of a stain for differentiation of gram-positive and gram-negative bacteria by Hans Christian Gram. The technique relies on the principle that pathogens have unique structural features, which can be recognized by specifically labeled organic molecules. A hundred years of extensive screening efforts led to discovery of a limited assortment of organic probes that are used for identification and differentiation of bacteria. A new challenge--continuous monitoring of biological threats--requires long lasting molecular probes capable of tight specific binding of pathogens in unfavorable conditions. To respond to the challenge, probe technology is being revolutionized by utilizing methods of combinatorial chemistry, phage display and directed molecular evolution. This review describes how molecular evolution methods are applied for development of peptide, antibody and phage probes, and summarizes the author's own data on development of landscape phage probes against Salmonella typhimurium. The performance of the probes in detection of Salmonella is illustrated by a precipitation test, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and fluorescent, optical and electron microscopy.

Antibodies↗
PubMed2002

De novo design of biocatalysts.

The challenging field of de novo enzyme design is beginning to produce exciting results. The application of powerful computational methods to functional protein design has recently succeeded at engineering target activities. In addition, efforts in directed evolution continue to expand the transformations that can be accomplished by existing enzymes. The engineering of completely novel catalytic activity requires traversing inactive sequence space in a fitness landscape, a feat that is better suited to computational design. Optimizing activity, which can include subtle alterations in backbone conformation and protein motion, is better suited to directed evolution, which is highly effective at scaling fitness landscapes towards maxima. Improved rational design efforts coupled with directed evolution should dramatically improve the scope of de novo enzyme design.

Antibodies, Catalytic↗
PubMed2005

Mimicking natural evolution in metallo-beta-lactamases through second-shell ligand mutations.

Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most beta-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.

Amino Acid Substitution↗
PubMed1993

An ankryin-like protein in ascidian eggs and its role in the evolution of direct development.

An erythrocyte anti-ankryin antibody was used to investigate the presence and distribution of ankryin in eggs and embryos of ascidian species with different modes of development. In eggs of the indirect developer Ascidia ceratodes, anti-ankryin reacted with a 210 kDa polypeptide which has an electrophoretic mobility similar to the vertebrate ankryins. Immunofluorescence microscopy showed that the ankryin-like protein is co-distributed with the myoplasm throughout development. It is restricted to a thin layer under the plasma membrane in unfertilised eggs, undergoes ooplasmic segregation to the posterior pole of the zygote after fertilisation, and is distributed to the tail muscle cells during cleavage and embryogenesis. After gastrulation and neurulation, lower levels of the ankryin-like protein, presumably of zygotic origin, were observed in brain cells and in the apical margin of epidermal cells. The ankryin-like protein was also localised in the myoplasm in eggs and embryos of another indirect developing species, Halocynthia roretzi. The ankryin-like protein may link the cytoskeleton with the plasma membrane in ascidian eggs, as it does in vertebrate erythrocytes. In contrast to A. ceratodes and H. roretzi, which are members of the families Ascidiidae and Pyuridae respectively, the pattern of ankryin-like protein expression was changed in five species in the family Molgulidae. These molgulid ascidians exhibit either indirect or direct development, and eggs of the direct developing species have lost or modified the myoplasm. The ankryin-like protein was present in young oocytes but failed to persist during oogenesis and disappeared in mature eggs and embryos of these molgulid species. The change in ankryin-like protein expression may be a preadaptation for loss of the myoplasm and the evolution of direct development.

Animals↗
PubMed2005

Simulation modeling of pooling for combinatorial protein engineering.

Pooling in directed-evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. The authors developed and validated a Monte Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using a simplified test system of 2 enzymes, betagalactosidase (supermutant, or greatly improved enzyme) and beta-glucuronidase (dud, or enzyme with ancestral level of activity), the model accurately predicted the number of supermutants detected in experiments within a factor of 2. Additional simulations using more complex activity distributions show the versatility of the model. Pooling is most suited to cases such as the directed evolution of new function in a protein, where the background level of activity is minimized, making it easier to detect small increases in activity level. Pooling is most successful when a sensitive assay is employed. Using the model will increase the throughput of screening procedures for directed-evolution experiments and thus lead to speedier engineering of proteins.

Cells, Cultured↗