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PubMed2006

Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution.

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.

Binding Sites↗
PubMed2000

Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine.

Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate. The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.

Amidohydrolases↗
PubMed2002

Genome shuffling of Lactobacillus for improved acid tolerance.

Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.

Catalysis↗
PubMed2004

A novel ADP- and zinc-binding fold from function-directed in vitro evolution.

A great challenge to biologists is to create proteins with novel folds and tailored functions. As an alternative to de novo protein design, we investigated the structure of a randomly generated protein targeted to bind ATP. The crystal structure reveals a novel alpha/beta fold bound to its ligand, representing both the first protein structure derived from in vitro evolution and the first nucleotide-binding protein stabilized by a zinc ion.

Adenosine Diphosphate↗
PubMed2007

Ribosome-inactivation display system.

We present a novel strategy for the connection of phenotype and genotype in vitro that can be used for the selection of functional proteins. The strategy involves the generation of a stable complex among a ribosome, an messenger RNA and its translated protein, without removal of the termination codon, as a result of the action of the ricin A chain during translation. The technique requires no transfection, no chemical synthesis, no ligation, and no removal of the termination codon. Thus, our novel ribosome-inactivation display system should provide, without loss of the pool population, a reliable, simple, and robust selection system for the in vitro evolution of the properties of proteins in a predictable direction by a combination of randomization and appropriate selection strategies.

Amino Acid Sequence↗
PubMed2006

Experimental evolution of resistance in Paramecium caudatum against the bacterial parasite Holospora undulata.

Host-parasite coevolution is often described as a process of reciprocal adaptation and counter adaptation, driven by frequency-dependent selection. This requires that different parasite genotypes perform differently on different host genotypes. Such genotype-by-genotype interactions arise if adaptation to one host (or parasite) genotype reduces performance on others. These direct costs of adaptation can maintain genetic polymorphism and generate geographic patterns of local host or parasite adaptation. Fixation of all-resistant (or all-infective) genotypes is further prevented if adaptation trades off with other host (or parasite) life-history traits. For the host, such indirect costs of resistance refer to reduced fitness of resistant genotypes in the absence of parasites. We studied (co)evolution in experimental microcosms of several clones of the freshwater protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. After two and a half years of culture, inoculation of evolved and naive (never exposed to the parasite) hosts with evolved and founder parasites revealed an increase in host resistance, but not in parasite infectivity. A cross-infection experiment showed significant host clone-by-parasite isolate interactions, and evolved hosts tended to be more resistant to their own (local) parasites than to parasites from other hosts. Compared to naive clones, evolved host clones had lower division rates in the absence of the parasite. Thus, our study indicates de novo evolution of host resistance, associated with both direct and indirect costs. This illustrates how interactions with parasites can lead to the genetic divergence of initially identical populations.

Animals↗
PubMed1997

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Carboxylic Ester Hydrolases↗
PubMed2002

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.

Alkaline Phosphatase↗
PubMed1996

Engineering new functions and altering existing functions.

Structural and mechanistic information, sequence comparisons, and site-directed mutagenesis data continue to provide a basis for the rational design of new protein functions and the alteration of existing functions. Random mutagenesis and 'directed evolution' approaches, however, are making significant headway in solving protein engineering problems, proving highly practical for tuning properties such as enzyme substrate specificity.

Binding Sites↗
PubMed2003

Microarray-based method for combinatorial library sequence mapping and characterization.

Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.

Base Sequence↗
PubMed1997

Continuous in vitro evolution of catalytic function.

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Base Sequence↗
PubMed2005

Identification of a carotenoid oxygenase synthesizing acyclic xanthophylls: combinatorial biosynthesis and directed evolution.

A carotenoid desaturase homolog from Staphylococcus aureus (CrtOx) was identified. When expressed in engineered E. coli cells synthesizing linear C(30) carotenoids, polar carotenoid products were generated, identified as aldehyde and carboxylic acid C(30) carotenoid derivatives. The major product in this engineered pathway is the fully desaturated C(30) dialdehyde carotenoid 4,4'-diapolycopen-4,4'-dial. Very low carotenoid yields were observed when CrtOx was complemented with the C(40) carotenoid lycopene pathway. But extension of an in vitro evolved pathway of the fully desaturated 2,4,2',4'-tetradehydrolycopene produced the structurally novel fully desaturated C(40) dialdehyde carotenoid 2,4,2',4'-tetradehydrolycopendial. Directed evolution of CrtOx by error-prone PCR resulted in a number of variants with higher activity on C(40) carotenoid substrates and improved product profiles. These findings may provide new biosynthetic routes to highly polar carotenoids with unique spectral properties desirable for a number of industrial and pharmaceutical applications.

Carotenoids↗
PubMed2004

Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase.

The Escherichia coli aspartate (AATase) and tyrosine (TATase) aminotransferases share 43% sequence identity and 72% similarity, but AATase has only 0.08% and 0.01% of the TATase activities (k(cat)/K(m)) for tyrosine and phenylalanine, respectively. Approximately 5% of TATase activity was introduced into the AATase framework earlier both by rational design (six mutations, termed HEX) and by directed evolution (9-17 mutations). The enzymes realized from the latter procedure complement tyrosine auxotrophy in TATase deficient E. coli. HEX complements even more poorly than does wild-type AATase, even though the (k(cat)/K(m)) value for tyrosine exhibited by HEX is similar to those of the enzymes found from directed evolution. HEX, however, is characterized by very low values of K(m) and K(D) for dicarboxylic ligands, and by a particularly slow release for oxaloacetate, the product of the reaction with aspartate and a TCA cycle intermediate. These observations suggest that HEX exists largely as an enzyme-product complex in vivo. HEX was therefore subjected to a single round of directed evolution with selection for complementation of tyrosine auxotrophy. A variant with a single amino acid substitution, A293D, exhibited substantially improved TATase function in vivo. The A293D mutation alleviates the tight binding to dicarboxylic ligands as K(m)s for aspartate and alpha-ketoglutarate are >20-fold higher in the HEX + A293D construct compared to HEX. This mutation also increased k(cat)/K(m)(Tyr) threefold. A second mutation, I73V, elicited smaller but similar effects. Both residues are in close proximity to Arg292 and the mutations may function to modulate the arginine switch mechanism responsible for dual substrate recognition in TATases and HEX.

Amino Acids↗
PubMed2004

Pure translation display.

Methods such as monoclonal antibody technology, phage display, and ribosome display provide genetic routes to the selection of proteins and peptides with desired properties. However, extension to polymers of unnatural amino acids is problematic because the translation step is always performed in vivo or in crude extracts in the face of competition from natural amino acids. Here, we address this restriction using a pure translation system in which aminoacyl-tRNA synthetases and other competitors are deliberately omitted. First, we show that such a simplified system can synthesize long polypeptides. Second, we demonstrate "pure translation display" by selecting from an mRNA library only those mRNAs that encode a selectable unnatural amino acid upstream of a peptide spacer sequence long enough to span the ribosome tunnel. Pure translation display should enable the directed evolution of peptide analogs with desirable catalytic or pharmacological properties.

Amino Acid Sequence↗
PubMed2005

Preparative expression of secreted proteins in bacteria: status report and future prospects.

The expression of heterologous secreted proteins in Escherichia coli is widely employed for laboratory and preparative purposes. Thanks to advances in expression technologies over the past 25 years, many mammalian proteins can now be produced routinely in secreted form with yields in the gram/litre scale. Nonetheless, ensuring efficient secretion across the inner membrane, and preventing proteolytic degradation, incorrect disulfide-bond formation and aggregation into periplasmic inclusion bodies, frequently presents significant challenges. Recent advances in the understanding of the periplasmic folding quality control system are leading to new strategies to facilitate the expression of heterologous secreted proteins. In parallel, protein design and directed evolution approaches are beginning to be exploited for engineering of the cellular protein folding machinery to achieve further improvements in protein expression.

Adenosine Triphosphatases↗
PubMed2005

Rapid creation of a novel protein function by in vitro coevolution.

We have developed a simple and efficient method for creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves design of a hypothetical pathway for the target function followed by stepwise directed evolution of the corresponding protein along the pathway. As a test case, this strategy was used to engineer variants of human estrogen receptor alpha ligand-binding domain (hERalphaLBD) with novel corticosterone activity. Two steroids, testosterone and progesterone, that provide a progressive structural bridge between 17beta-estradiol and corticosterone, were chosen to assist the directed evolution of hERalphaLBD. A total of approximately 10(6) variants were screened in four rounds of random mutagenesis, resulting in two hERalphaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches and also shed light on the natural evolution of nuclear hormone receptors. This in vitro coevolution approach should provide a powerful, broadly applicable tool for engineering biological molecules and systems with novel functions.

Corticosterone↗
PubMed2004

An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases.

Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT(0)-ACP(0) was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT(0)-ACP(0) fragments, which were used to replace sacB by lambda-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.

Acyltransferases↗
PubMed2006

Reconstruction of functional beta-propeller lectins via homo-oligomeric assembly of shorter fragments.

The modular nature of protein folds suggests that present day proteins evolved via duplication and recombination of smaller functional elements. However, the reconstruction of these putative evolutionary pathways after many millions of years of evolutionary drift has thus far proven difficult, with all attempts to date failing to produce a functional protein. Tachylecin-2 is a monomeric 236 amino acid, five-bladed beta-propeller with five sugar-binding sites. This protein was isolated from a horseshoe crab that emerged ca 500 million years ago. The modular, yet ancient, nature of Tachylectin-2 makes it an excellent model for exploring the evolution of proteins from smaller subunits. To this end, we generated genetically diverse libraries by incremental truncation of the Tachylectin-2 gene and screened them for functional lectins. A number of approximately 100 amino acid residue segments were isolated with the ability to assemble into active homo-pentamers. The topology of most of these segments follows a "hidden" module that differs from the modules observed in wild-type Tachylectin-2, yet their biophysical properties and sugar binding activities resemble the wild-type's. Since the pentamer's molecular mass is twofold higher than the wild-type (approximately 500 amino acid residues), the structure of these oligomeric forms is likely to also differ. Our laboratory evolution experiments highlight the versatility and modularity of the beta-propeller fold, while substantiating the hypothesis that proteins with high internal symmetry, such as beta-propellers, evolved from short, functional gene segments that, at later stages, duplicated, fused, and rearranged, to yield the folds we recognise today.

Amino Acid Sequence↗