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Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability.

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.

Agrobacterium tumefaciens↗
PubMed2003

Preferred RNA binding sites for a threading intercalator revealed by in vitro evolution.

In pursuit of small molecules capable of controlling the function of RNA targets, we have explored the RNA binding properties of peptide-acridine conjugates (PACs). In vitro evolution (SELEX) was used to isolate RNAs capable of binding the PAC Ser-Val-Acr-Arg, where Acr is an acridine amino acid. The PAC binds RNA aptamers selectively and with a high degree of discrimination over DNA. PAC binding sites contain the base-paired 5'-CpG-3' sequence, a known acridine intercalation site. However, RNA structure flanking this sequence causes binding affinities to vary over 30-fold. The preferred site (K(D) = 20 nM) contains a base-paired 5'-CpG-3' step flanked on the 5' side by a 4 nt internal loop and the 3' side by a bulged U. Several viral 5'- and 3'-UTR RNA sequences that likely form binding sites for this PAC are identified.

Acridines↗
PubMed2006

Metabolic analysis of adaptive evolution for in silico-designed lactate-producing strains.

Experimental evolution is now frequently applied to many biological systems to achieve desired objectives. To obtain optimized performance for metabolite production, a successful strategy has been recently developed that couples metabolic engineering techniques with laboratory evolution of microorganisms. Previously, we reported the growth characteristics of three lactate-producing, adaptively evolved Escherichia coli mutant strains designed by the OptKnock computational algorithm. Here, we describe the use of (13)C-labeled experiments and mass distribution measurements to study the evolutionary effects on the fluxome of these differently designed strains. Metabolic flux ratios and intracellular flux distributions as well as physiological data were used to elucidate metabolic responses over the course of adaptive evolution and metabolic differences among strains. The study of 3 unevolved and 12 evolved engineered strains as well as a wild-type strain suggests that evolution resulted in remarkable improvements in both substrate utilization rate and the proportion of glycolytic flux to total glucose utilization flux. Among three strain designs, the most significant increases in the fraction of glucose catabolized through glycolysis (>50%) and the glycolytic fluxes (>twofold) were observed in phosphotransacetylase and phosphofructokinase 1 (PFK1) double deletion (pta- pfkA) strains, which were likely attributed to the dramatic evolutionary increase in gene expression and catalytic activity of the minor PFK encoded by pfkB. These fluxomic studies also revealed the important role of acetate synthetic pathway in anaerobic lactate production. Moreover, flux analysis suggested that independent of genetic background, optimal relative flux distributions in cells could be achieved faster than physiological parameters such as nutrient utilization rate.

Acetate-CoA Ligase↗
PubMed2000

Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif.

We report the evolution of an RNA aptamer to change its binding specificity. RNA aptamers that bind the free amino acid tyrosine were in vitro selected from a degenerate pool derived from a previously selected dopamine aptamer. Three independent sequences bind tyrosine in solution, the winner of the selection binding with a dissociation constant of 35 microM. Competitive affinity chromatography with tyrosine-related ligands indicated that the selected aptamers are highly L-stereo selective and also recognize L-tryptophan and L-dopa with similar affinity. The binding site was localized by sequence comparison, analysis of minimal boundaries, and structural probing upon ligand binding. Tyrosine-binding sites are characterized by the presence of both tyrosine (UAU and UAC) and termination (UAG and UAA) triplets.

Base Sequence↗
PubMed2006

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging.

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

Animals↗
PubMed1998

Towards the design of rare cutting restriction endonucleases: using directed evolution to generate variants of EcoRV differing in their substrate specificity by two orders of magnitude.

The restriction endonuclease EcoRV cleaves DNA highly specifically within GATATC sequences. In order to create EcoRV variants that have an extended recognition site we have employed a semi-rational random mutagenesis/selection procedure. Twenty-two amino acid residues were subjected to random mutagenesis and about 500 EcoRV variants representing three generations of mutants were screened. Among these some highly active variants that strongly prefer AT-flanked cleavage sites (e.g. S183A/Q224R, T93S/I103F/S183A/T222S or N97T/S183A/T222S) and others that prefer GC flanks (e.g. K104N/A181T) were identified. As wild-type EcoRV does not discriminate between these cleavage sites, the generation of these variants represents a significant first step towards redesigning EcoRV to become an 8 or 10 bp cutter. Such enzymes, only very rarely found in nature, could be extremely helpful for the manipulation of large DNA fragments.

Deoxyribonucleases, Type II Site-Specific↗
PubMed2006

Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide.

In two cycles of an error-prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4-fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.

Acrylic Resins↗
PubMed2000

Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution.

Combinatorial library selections through the systematic evolution of ligands by exponential enrichment (SELEX) technique identify so-called nucleic acid aptamers that bind with high-affinity and specificity to a wide range of selected molecules. However, the modest chemical functionality of nucleic acids poses some limits on their versatility as binders and catalysts, and, furthermore, the sensitivity of pure RNA- and DNA-based aptamers to nucleases restricts their use as therapeutic and diagnostic agents. Here we review synthetic chemistries for modifying nucleotides that have been developed to enhance the affinity of aptamers for targets and to increase their stability in biological fluids. Implementation of in vitro selections with modified nucleotides promises to be an elegant technique for the creation of ligands with novel physical and chemical properties and is anticipated to have a significant impact on biotechnology, diagnostics and drug development. The current molecular designs and applications of modified nucleotides for in vitro selections are reviewed, along with a discussion of future developments expected to further the utility of this approach in both practical and theoretical terms.

Animals↗
PubMed2006

Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase.

Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.

Aldehyde-Lyases↗
PubMed2003

Directed evolution of a single-chain class II MHC product by yeast display.

Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7). The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7). A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.

Amino Acid Sequence↗
PubMed2006

In vitro evolution of flexizymes that function under the conditions in translation system.

Flexizyme is an artificial ribozyme capable of acylating any kinds of tRNAs with virtually no limitation of acid donor substrates. Although the flexizyme system is a highly flexible tRNA acylation tool, it requires relatively high Mg(2+) concentrations (>50 mM) for the full function. For instance, lowering the Mg(2+) concentration to 10 mM, where the in vitro translation system generally functions, resulted in significant decrease in activity. To overcome this limitation, we implanted a new random domain into a part of flexizyme to aim at selecting a new functional domain that would exhibit acylation activity at lower Mg(2+) concentrations. Indeed, in vitro selection of active species from such an RNA pool afforded a new flexizyme, called mdFx, showing a 3-fold higher yield at 10 mM Mg(2+) concentration. Thus, this new flexizyme opens a new avenue to construct an mdFx-transcription-translation coupled system.

Base Sequence↗
PubMed2006

[Directed evolution of Thermophilic esterase from the archaeon Aeropyrum pemix K1].

Thermophilic esterase (APE1547) from Aeropyrum pernix K1 was subjected to error-prone PCR (epPCR) to enhance activity. For the screening of mutants, an efficient and reliable assay suitable for high throughput screening was developed based on the enzyme thermostability. Two successive rounds of random mutagenesis by epPCR resulted in a four amino acid substitution variant M020 with significantly increased activity (six-fold under the screening condition. Further assay for the purified enzymes showed that the mutant possess 1.5-fold higher specific activity and nearly 4-fold higher expressed level than the wild-type. The mutant has an optimal activity at pH 8.5, corresponding to an alkaline shift of 0.5 pH unit compared to the wild type. The structure analysis suggests that R526S may contribute to the enhanced activity and the shift of pKi.

Aeropyrum↗
PubMed2001

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Base Sequence↗
PubMed1999

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

Alleles↗
PubMed2005

Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones.

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant omega-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, omega-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using omega-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.

Amines↗
PubMed2000

Modeling DNA mutation and recombination for directed evolution experiments.

Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data.

Animals↗
PubMed1998

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Binding Sites↗
PubMed2005

Synthesis of screening substrates for the directed evolution of sialic acid aldolase: towards tailored enzymes for the preparation of influenza A sialidase inhibitor analogues.

The stereoselective synthesis of two epimeric screening substrates, (4R, 5R, 6R)- and (4S, 5R, 6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxy-hexanoic acid, for the directed evolution of sialic acid aldolase is described. The complementary methods relied on stereoselective indium-mediated additions of ethyl alpha-bromomethyl acrylate to functionalised aldehydes. With an alpha-hydroxy aldehyde, (2R, 3R)-2,3-dihydroxy-4-oxo butanoic acid dipropylamide, the addition was chelation controlled, and the syn product, (6R, 5R, 4S)-6-dipropylcarbamoyl-2-methylidene-4,5,6-trihydroxy-hexanoic acid ethyl ester, was obtained. In contrast, the stereochemical outcome of the addition to (2R, 3R)-N,N-dipropyl-2,3-O-isopropylidene-4-oxobutyramide was consistent with Felkin-Anh control, and the anti adduct, (4R, 5R, 6R)-6-dipropylcarbamoyl-2-methylidene-4-hydroxy-5,6-O-isopropylidene-hexanoic acid ethyl ester, was the major product. Ozonolysis and deprotection gave the screening substrates as mixtures of furanose and pyranose forms, in good yields.

Directed Molecular Evolution↗