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PubMed2005

Combinatorial library approaches for improving soluble protein expression in Escherichia coli.

High-throughput screening methodologies are already used in structural biology to define efficient protein crystallization and expression conditions. Recently, screening approaches have been extended to the optimization of genetic constructs for improved soluble protein expression. With similarities to the directed evolution strategies used in protein engineering, a target gene encoding a poorly expressed protein is mutated by truncation, fragmentation or point mutation. Rare clones with improved protein expression characteristics are then isolated from the random library using a phenotypic screen or selection. This article reviews the progress in this field and provides a general overview of relevant mutation methods, screens and selections.

Animals↗
PubMed2006

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale.

We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.

Adaptation, Physiological↗
PubMed1993

Directional mutation pressure, mutator mutations, and dynamics of molecular evolution.

Using a general form of the directional mutation theory, this paper analyzes the effect of mutations in mutator genes on the G+C content of DNA, the frequency of substitution mutations, and evolutionary changes (cumulative mutations) under various degrees of selective constraints. Directional mutation theory predicts that when the mutational bias between A/T and G/C nucleotide pairs is equilibrated with the base composition of a neutral set of DNA nucleotides, the mutation frequency per gene will be much lower than the frequency immediately after the mutator mutation takes place. This prediction explains the wide variation of the DNA G+C content among unicellular organisms and possibly also the wide intragenomic heterogeneity of third codon positions for the genes of multicellular eukaryotes. The present analyses lead to several predictions that are not consistent with a number of the frequently held assumptions in the field of molecular evolution, including belief in a constant rate of evolution, symmetric branching of phylogenetic trees, the generality of higher mutation frequency for neutral sets of nucleotides, the notion that mutator mutations are generally deleterious because of their high mutation rates, and teleological explanations of DNA base composition.

Animals↗
PubMed2005

Directed evolution of metabolic pathways.

The modification of cellular metabolism is of biotechnological and commercial significance because naturally occurring metabolic pathways are the source of diverse compounds used in fields ranging from medicine to bioremediation. Directed evolution is the experimental improvement of biocatalysts or cellular properties through iterative genetic diversification and selection procedures. The creation of novel metabolic functions without disrupting the balanced intracellular pool of metabolites is the primary challenge of pathway manipulation. The introduction of coordinated changes across multiple genetic elements, in conjunction with functional selection, presents an integrated approach for the modification of metabolism with benign physiological consequences. Directed evolution formats take advantage of the dynamic structures of genomes and genomic sub-structures and their ability to evolve in multiple directions in response to external stimuli. The elucidation, design and application of genome-restructuring mechanisms are key elements in the directed evolution of cellular metabolic pathways.

Biotechnology↗
PubMed1999

Novel approaches for discovering industrial enzymes.

New technologies for enzyme discovery are changing the rules of the game for industrial biocatalysis. More kinds of enzymes are available, their hardiness is increasing, and their costs are coming down. These changes are the key drivers for a rebirth of interest in industrial applications of enzymes. The major enabling discovery approaches include screening of biodiversity, genomic sequencing, directed evolution and phage display.

Directed Molecular Evolution↗
PubMed2006

Recent advances in biocatalysis by directed enzyme evolution.

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes recent advances in applying directed evolution approaches to alter various enzyme properties such as activity, selectivity (enantio- and regio-), substrate specificity, stability, and solubility. Special attention will be paid to the creation of novel enzyme activities and products by directed evolution.

Bacteria↗
PubMed2025

Bacterial directed evolution of CRISPR base editors.

Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.

Gene Editing↗
PubMed2004

Fluorescent intensity of a novel NADPH-binding protein of Vibrio vulnificus can be improved by directed evolution.

Blue fluorescent protein, BfgV, found from Vibrio vulnificus CKM-1, fluoresces through augmenting the intrinsic fluorescence of bound NADPH. Random mutagenesis and DNA shuffling were applied to increase the fluorescent intensity of BfgV. The wild type bfgV gene was subjected to four cycles of mutagenesis processes. A prominent D7 mutant protein had fluorescent intensity four times larger than wild type BfgV. The emission wavelength of this mutant protein appeared at 440 nm, which was 16 nm shorter than that of BfgV. There were eight amino acid substitutions in D7. As these substitutions were assigned to the modeled 3D structure of BfgV, three of them, V83M, G176S, and E179K, were shown to be located around NADPH-binding site. Time course analysis indicated the synthesis of D7 protein and fluorescent expression in Escherichia coli transformants were synchronic. This property was different from that of wild type GFP.

Amino Acid Sequence↗
PubMed2000

Enantioselective enzymes for organic synthesis created by directed evolution.

A new concept for the creation of enzymes displaying improved enantioselectivity in a given reaction is described; it is based on "evolution in the test tube". Accordingly, proper molecular biological methods for random mutagenesis, gene expression, and high-throughput screening systems for the rapid assay of enantioselectivity are combined. Several rounds of mutagenesis and screening are generally necessary in order to create mutant enzymes that show high degrees of enantioselectivity, as in the case of the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2 % ee (E = 1) increases to > 90% ee (E = 25).

Catalysis↗
PubMed2004

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Bacteria↗
PubMed2005

The first characterisation of the overall variability of repetitive units in a species reveals unexpected features of satellite DNA.

We investigated the overall variability of the S1a satellite DNA repeats in ten European populations of Rana temporaria by a new procedure that determines the average sequence of the repeats in a genome. The average genomic sequences show that only 17% of the S1a repeat sequence (494 bp) is variable. The variable positions contain the same major and minor bases in all or many of the population samples tested, but the percentages of these bases can greatly vary among populations. This indicates the presence in the species of an enormous number of repeats having a different distribution of bases in these variable positions. Individual genomes contain thousands of repeat variants, but these mixtures have very similar characteristics in all populations because they present the same type of restricted and species-specific variability. Southern blots analyses and sequences of cloned S1a repeats fully support this conclusion. The S1 satellite DNA of other European brown frog species also presents properties indicating the same type of variability. This first characterisation of the overall repeat variability of a satellite DNA in a species has revealed features that cannot be determined by gene conversion and crossing over. Our results suggest that a specific directional process based on rolling circle amplification should play a relevant role in the evolution of satellite DNA.

Animals↗
PubMed2000

Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution.

The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme. Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 degrees C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold. In most cases, this was achieved at the expense of beta-galactosidase activity at 20 degrees C and total catalytic efficiency at 90 degrees C. Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site. The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl-beta-D-galactopyra noside hydrolysis. This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates.

Amino Acid Sequence↗
PubMed2005

Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions.

The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.

Bacillus subtilis↗
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↗
PubMed1999

An approach to optimizing the active site in a glutathione transferase by evolution in vitro.

A glutathione transferase (GST) mutant with four active-site substitutions (Phe(10)-->Pro/Ala(12)-->Trp/Leu(107)-->Phe/Leu(108)-->Arg) (C36) was isolated from a library of active-site mutants of human GST A1-1 by the combination of phage display and mechanism-based affinity adsorption [Hansson, Widersten and Mannervik (1997) Biochemistry 36, 11252-11260]. C36 was selected on the basis of its affinity for the transition-state analogue 1-(S-glutathionyl)-2,4, 6-trinitrocyclohexadienate. C36 affords a 10(5)-fold rate enhancement over the uncatalysed reaction between reduced glutathione and 1-chloro-2,4-dinitrobenzene (CDNB), as evidenced by the ratio between k(cat)/K(m) and the second-order rate constant k(2). The present study shows that C36 can evolve to an even higher catalytic efficiency by an additional site-specific mutation. Random mutations of the fifth active-site residue 208 allowed the identification of 18 variants, of which the mutant C36 Met(208)-->Cys proved to be the most active form. The altered activity was substrate selective such that the catalytic efficiency with CDNB and with 1-chloro-6-trifluoromethyl-2,4-dinitrobenzene were increased 2-3-fold, whereas the activity with ethacrynic acid was decreased by a factor of 8. The results show that a single-point mutation in the active site of an enzyme may modulate the catalytic activity without being directly involved as a functional group in the enzymic mechanism. Such limited modifications are relevant both to the natural evolution and the in vitro redesign of proteins for novel functions.

Amino Acid Substitution↗
PubMed2003

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution.

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.

Amino Acid Oxidoreductases↗