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PubMed2007

A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability.

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such proteins, but the results to date have been of mixed success. Here, we present a robust method involving the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally, selection at physiological temperatures, to generate an enzyme with improved stability. Three cycles of directed evolution comprising of random mutagenesis, DNA shuffling, and selection at 37 degrees C were used, using the bacterial enzyme TEM-1 beta-lactamase as a model protein to yield deletion mutants with in vivo ampicillin resistance levels comparable to wild-type (wt) enzyme. Kinetic studies demonstrate the selected mutant to have a significantly improved thermostability relative to its wt counterpart. Elongation of this mutant to the full-length gene resulted in a beta-lactamase variant with dramatically increased thermostability. This technique was so fruitful that the evolved enzyme retained its maximum catalytic activity even 20 degrees C above its wt parent protein optimum. Thus, structural perturbation by terminal truncation and subsequent compensation by directed evolution at physiological temperatures is a fast, efficient, and highly effective way to improve the thermostability of proteins without the need for selecting at elevated temperatures.

Amino Acid Sequence↗
PubMed2001

Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes.

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.

Allosteric Regulation↗
PubMed2007

Novel enzymes through design and evolution.

The generation of enzymes with new catalytic activities remains a major challenge. So far, several different strategies have been developed to tackle this problem, including site-directed mutagenesis, random mutagenesis (directed evolution), antibody catalysis, computational redesign, and de novo methods. Using these techniques, a broad array of novel enzymes has been created (aldolases, decarboxylases, dehydratases, isomerases, oxidases, reductases, and others), although their low efficiencies (10 to 100 M(-1) s(-l)) compared to those of the best natural enzymes (10(6) to 10(8) M(-1) s(-1)) remains a significant concern. Whereas rational design might be the most promising and versatile approach to generating new activities, directed evolution seems to be the best way to optimize the catalytic properties of novel enzymes. Indeed, impressive successes in enzyme engineering have resulted from a combination of rational and random design.

Catalysis↗
PubMed2000

Directed evolution study of temperature adaptation in a psychrophilic enzyme.

We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C). The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins.

Adaptation, Physiological↗
PubMed2004

Metabolic gene-deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes.

Genome-scale metabolic models have a promising ability to describe cellular phenotypes accurately. Here we show that strains of Escherichia coli carrying a deletion of a single metabolic gene increase their growth rates (by 87% on average) during adaptive evolution and that the endpoint growth rates can be predicted computationally in 39 of 50 (78%) strains tested. These results show that computational models can be used to predict the eventual effects of genetic modifications.

Directed Molecular Evolution↗
PubMed2003

A modular, bifunctional RNA that integrates itself into a target RNA.

Nature often combines independent functional domains to achieve complex function, but this approach has not been extensively explored with artificial enzymes. Here, a group I ribozyme, which can act as an endoribonuclease, was partnered with the R3C ribozyme, which catalyzes the ligation of RNA molecules. The conjoined ribozymes have the potential to perform successive RNA cleavage and joining reactions, resulting in their mutual integration into a target RNA substrate. When simply joined together, however, the ribozymes were unable to achieve this outcome because of inefficient transfer of the substrate between the two catalytic subunits. In vitro evolution was used to optimize the behavior of the conjoined ribozymes, resulting in bifunctional molecules with substantially improved integration activity. The ligase subunit of these molecules was unchanged, whereas the group I subunit acquired several mutations, mostly in peripheral regions. The generation and study of this bifunctional assembly helps shed light on the evolution of modular enzymes and the obstacles that must be overcome in bringing together independent functional domains. These molecules also may be useful as tools for the insertional mutagenesis of target mRNAs.

Base Sequence↗
PubMed2001

Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution.

We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli. The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme. Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.

Amino Acid Motifs↗
PubMed2005

High-throughput screens and selections of enzyme-encoding genes.

The availability of vast gene repertoires from both natural sources (genomic and cDNA libraries) and artificial sources (gene libraries) demands the development and application of novel technologies that enable the screening or selection of large libraries for a variety of enzymatic activities. We describe recent developments in the selection of enzyme-coding genes for directed evolution and functional genomics. We focus on HTS approaches that enable selection from large libraries (>10(6) gene variants) with relatively humble means (i.e. non-robotic systems), and on in vitro compartmentalization in particular.

Animals↗
PubMed2006

Evolution of new protein topologies through multistep gene rearrangements.

New protein folds have emerged throughout evolution, but it remains unclear how a protein fold can evolve while maintaining its function, particularly when fold changes require several sequential gene rearrangements. Here, we explored hypothetical evolutionary pathways linking different topological families of the DNA-methyltransferase superfamily. These pathways entail successive gene rearrangements through a series of intermediates, all of which should be sufficiently active to maintain the organism's fitness. By means of directed evolution, and starting from HaeIII methyltransferase (M.HaeIII), we selected all the required intermediates along these paths (a duplicated fused gene and duplicates partially truncated at their 5' or 3' coding regions) that maintained function in vivo. These intermediates led to new functional genes that resembled natural methyltransferases from three known classes or that belonged to a new class first seen in our evolution experiments and subsequently identified in natural genomes. Our findings show that new protein topologies can evolve gradually through multistep gene rearrangements and provide new insights regarding these processes.

Amino Acid Sequence↗
PubMed2001

Protease inhibitors and directed evolution: enhancing plant resistance to nematodes.

Plant nematodes are agricultural pests, the control of which relies on chemical nematicides and fumigants that are among the most toxic and environmentally damaging of all agrochemicals. New approaches to control, based on transgenic resistance, would provide important health and environmental benefits. In this chapter we consider briefly some targets for engineering nematode resistance and discuss the use of plant protease inhibitors as anti-feedants. This approach has provided plants that display good levels of resistance against a range of nematode species. To enhance this defence strategy further we are investigating the value of directed evolution to improve the characteristics of protease inhibitors. We describe the approaches of DNA shuffling and phage display that are being used to create and screen variant libraries in the search for inhibitors with improved features.

Animals↗
PubMed2000

Reciprocal domain evolution within a transactivator in a restricted sequence space.

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Amino Acid Sequence↗
PubMed2004

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity.

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme trade mark, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.

Catalysis↗
PubMed2003

Directed evolution of a yeast arsenate reductase into a protein-tyrosine phosphatase.

Arsenic, which is ubiquitous in the environment and comes from both geochemical and anthropogenic sources, has become a worldwide public health problem. Every organism studied has intrinsic or acquired mechanisms for arsenic detoxification. In Saccharomyces cerevisiae arsenate is detoxified by Acr2p, an arsenate reductase. Acr2p is not a phosphatase but is a homologue of CDC25 phosphatases. It has the HCX5R phosphatase motif but not the glycine-rich phosphate binding motif (GXGXXG) that is found in protein-tyrosine phosphatases. Here we show that creation of a phosphate binding motif through the introduction of glycines at positions 79, 81, and 84 in Acr2p resulted in a gain of phosphotyrosine phosphatase activity and a loss of arsenate reductase activity. Arsenate likely achieved geochemical abundance only after the atmosphere became oxidizing, creating pressure for the evolution of an arsenate reductase from a protein-tyrosine phosphatase. The ease by which an arsenate reductase can be converted into a protein-tyrosine phosphatase supports this hypothesis.

Arsenate Reductases↗
PubMed2005

Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: production of an efficient enzyme for bioconversion of fine chemicals.

The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.

Amino Acid Sequence↗
PubMed2007

Droplets as microreactors for high-throughput biology.

Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.

Bacteria↗
PubMed2006

Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins.

Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.

Amino Acid Sequence↗
PubMed1997

Molding a peptide into an RNA site by in vivo peptide evolution.

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single alpha-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage lambda N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial alpha-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an "RNA world."

Amino Acid Sequence↗