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PubMed2002

eCodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments.

We present a systematic computational framework, eCodonOpt, for designing parental DNA sequences for directed evolution experiments through codon usage optimization. Given a set of homologous parental proteins to be recombined at the DNA level, the optimal DNA sequences encoding these proteins are sought for a given diversity objective. We find that the free energy of annealing between the recombining DNA sequences is a much better descriptor of the extent of crossover formation than sequence identity. Three different diversity targets are investigated for the DNA shuffling protocol to showcase the utility of the eCodonOpt framework: (i) maximizing the average number of crossovers per recombined sequence; (ii) minimizing bias in family DNA shuffling so that each of the parental sequence pair contributes a similar number of crossovers to the library; and (iii) maximizing the relative frequency of crossovers in specific structural regions. Each one of these design challenges is formulated as a constrained optimization problem that utilizes 0-1 binary variables as on/off switches to model the selection of different codon choices for each residue position. Computational results suggest that many-fold improvements in the crossover frequency, location and specificity are possible, providing valuable insights for the engineering of directed evolution protocols.

Aldose-Ketose Isomerases↗
PubMed2006

Genome-wide detection of polymorphisms at nucleotide resolution with a single DNA microarray.

A central challenge of genomics is to detect, simply and inexpensively, all differences in sequence among the genomes of individual members of a species. We devised a system to detect all single-nucleotide differences between genomes with the use of data from a single hybridization to a whole-genome DNA microarray. This allowed us to detect a variety of spontaneous single-base pair substitutions, insertions, and deletions, and most (>90%) of the approximately 30,000 known single-nucleotide polymorphisms between two Saccharomyces cerevisiae strains. We applied this approach to elucidate the genetic basis of phenotypic variants and to identify the small number of single-base pair changes accumulated during experimental evolution of yeast.

Directed Molecular Evolution↗
PubMed2004

DARTs: A DNA-based in vitro polypeptide display technology.

Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution. Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules. The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems. We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification. Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate. Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides. Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction. We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.

Base Sequence↗
PubMed2001

Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution.

Alpha-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.

Carbohydrate Conformation↗
PubMed2004

Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications.

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a "Darwinistic" process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.

Directed Molecular Evolution↗
PubMed2004

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents.

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.

Acetonitriles↗
PubMed2004

SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription.

Protein-primed replication of hepatitis B viruses (HBVs) is initiated by the chaperone dependent binding of the reverse transcriptase (P protein) to the bulged epsilon stem-loop on the pregenomic RNA, and the epsilon-templated synthesis of the 5' terminal nucleotides of the first DNA strand. How P protein recognizes the initiation site is poorly understood. In mammalian HBVs and in duck HBV (DHBV) the entire stem-loop is extensively base paired; in other avian HBVs the upper stem regions have a low base pairing potential. Initiation can be reconstituted with in vitro translated DHBV, but not HBV, P protein and DHBV epsilon (Depsilon) RNA. Employing the SELEX method on a constrained library of Depsilon upper stem variants, we obtained a series of well-binding aptamers. Most contained C-rich consensus motifs with very low base pairing potential; some supported initiation, others did not. Consensus-based secondary mutants allowed to pin down this functional difference to the residues flanking the conserved loop, and an unpaired U. In vitro active consensus sequences also supported virus replication. Hence, most of the upper stem acts as a spacer, which, if not base paired, warrants accessibility of relevant anchor residues. This suggests that the base paired Depsilon represents an exceptional rather than a prototypic avian HBV epsilon signal, and it offers an explanation as to why attempts to in vitro reconstitute initiation with human HBV have thus far failed.

Animals↗
PubMed2005

In vitro selection of high temperature Zn(2+)-dependent DNAzymes.

In vitro selection of Zn(2+)-dependent RNA-cleaving DNAzymes with activity at 90 degrees C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn(2+) over Pb(2+), Ca(2+), Cd(2+), Co(2+), Hg(2+), and Mg(2+). The Zn(2+)-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn(2+)-binding dissociation constant of 234 muM and that its activity increases with increasing temperatures from 50-90 degrees C. A fit of the Arrhenius plot data gave E(a) = 15.3 kcal mol(-1). Surprisingly, the selected Zn(2+)-dependent DNAzymes showed only a modest (approximately 3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn(2+) is present. Since this effect is highly specific for Zn(2+), this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.

Base Sequence↗
PubMed2003

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures.

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.

Biotechnology↗
PubMed2001

Tailoring in vitro evolution for protein affinity or stability.

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (k(off) at 4 degrees C of 10(-4) s(-1)) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible.

Amino Acid Sequence↗
PubMed2006

Making glucose oxidase fit for biofuel cell applications by directed protein evolution.

Progress in miniature chip-design raises demands for implantable power sources in health care applications such as continuous glucose monitoring of diabetic patients. Pioneered by Adam Heller, miniaturized enzymatic biofuel cells (mBCs) convert blood sugars into electrical energy by employing for example glucose oxidase (GOx) on the anode and bilirubin oxidase on the cathode. To match application demands it is crucial to increase lifetime and power output of mBCs. The power output has been limited by the performance of GOx on the anode. We developed a glucose oxidase detection assay (GODA) as medium-throughput screening system for improving GOx properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation which is recorded at 340 nm. The main advantage of the assay is that it detects the production of d-gluconolactone instead of the side-product hydrogen peroxide and enables to improve bioelectrochemical properties of GOx. For validating the screening system, a mutagenic library of GOx from Aspergillus niger (EC 1.1.3.4) was generated and screened for improved activity using Saccharomyces cerevisiae as host. Directed evolution resulted in a GOx mutant I115V with 1.4-1.5-fold improved activity for beta-d-glucose (Vmax from 7.94 to 10.81 micromol min(-1) mg(-1); Km approximately 19-21 mM) and oxygen consumption kinetics correlate well [Vmax (O2) from 5.94 to 8.34 micromol min(-1) mg(-1); Km (O2) from 700 to 474 microM]. The developed mutagenic protocol and GODA represent a proof-of-principle that GOx can be evolved by directed evolution in S. cerevisiae for putative use in biofuel cells.

Bioelectric Energy Sources↗
PubMed2000

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold.

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.

Aldose-Ketose Isomerases↗
PubMed2006

Directed evolution of adeno-associated virus yields enhanced gene delivery vectors.

Adeno-associated viral vectors are highly safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport and infection of resistant cell types. Changes must be made to the viral capsid to overcome these problems; however, very often insufficient information is available for rational design of improvements. We therefore applied a directed evolution approach involving the generation of large mutant capsid libraries and selection of adeno-associated virus (AAV) 2 variants with enhanced properties. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties.

Adenoviridae↗