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PubMed2005

Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency.

To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k(cat)/K(m)) for D-xylose and L-sorbose. P2OxB1 was provided with a C-terminal His(6)-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k(cat)/K(m) for D-glucose (5.3-fold), methyl-beta-D-glucoside (2.0-fold), D-galactose (4.8-fold), D-xylose (59.9-fold), and L-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of L-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.

Amino Acid Substitution↗
PubMed2002

In vitro evolution of amphioxus insulin-like peptide to mammalian insulin.

By site-directed mutagenesis, six insulin residues related to the insulin-receptor interaction were grafted, partially or fully, onto the corresponding position of a recombinant amphioxus insulin-like peptide (ILP) that contained the A- and B-domains of the deduced amphioxus ILP. After fermentation, purification, and enzymatic cleavage, six insulin-like double-chain ILP analogues were obtained: [A2Ile]ILP, [B12Val, B16Tyr]ILP, [B25Phe]ILP, [A2Ile, B12Val, B16Tyr, B25Phe]ILP (four-mutated ILP), [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe]ILP (five-mutated ILP), and [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr]ILP (six-mutated ILP). Circular dichroism analysis showed that such replacement did not significantly affect their secondary and tertiary structure compared with that of the wild-type ILP. The insulin-receptor-binding activity of the four-, five-, and six-mutated ILP was 0.14%, 11%, and 11% of native insulin, respectively; the other three ILP analogues acquired none of the detectable insulin-receptor-binding potency. The growth-promoting activities of the five- and six-mutated ILP were both about 50% of native insulin, while that of the wild-type ILP was not detectable. By structure-function-based mutagenesis, the completely inactive amphioxus ILP was converted into a molecule with moderate mammalian insulin activity. These results indicated the following: first, the grafted as well as those inborn insulin-receptor-binding related residues can form an insulin-receptor-binding patch on the ILP analogues; second, the ILP can be used as a scaffold molecule to investigate the role of the insulin residues; third, the natural evolution of amphioxus ILP to mammalian insulin is a possible process and can be mimicked in the laboratory.

Amino Acid Sequence↗
PubMed2005

Directed coevolution of stability and catalytic activity in calcium-free subtilisin.

We have coevolved high activity and hyperstability in subtilisin by sequentially randomizing 12 amino acid positions in calcium-free subtilisin. The optimal amino acid for each randomized site was chosen based on stability and catalytic properties and became the parent clone for the next round of mutagenesis. Together, the 12 selected mutations increased the half-life of calcium-free subtilisin at elevated temperature by 15,000-fold. The catalytic properties of the mutants were examined against a range of substrates. In general, only mutations occurring at or near the substrate-binding surface have measurable effects on catalytic constants. No direct influence of stability on catalytic properties was observed. A high-stability mutant, Sbt140, was a more efficient enzyme in terms of k(cat)/K(m) than a commercial version of subtilisin across a range of substrates but had a lower k(cat) against tight-binding substrates. The reason for this behavior was discerned by examining microscopic rate constants for the hydrolysis of a tight-binding peptide substrate. Burst kinetics were observed for this substrate, indicating that acylation is not rate-limiting. Although acylation occurs at the rate of substrate binding, k(cat) is attenuated by the slow release of the N-terminal product. Natural evolution appears to have optimized catalytic activity against a range of sequences by achieving a balance between substrate binding and the rate of release of the N-terminal product.

Acylation↗
PubMed2003

Controlling chirality.

New strategies are continually being developed for using enzymes to efficiently catalyse the enantioselective synthesis of chiral non-racemic compounds. Alternatives to asymmetric synthesis or kinetic resolution include dynamic kinetic resolution, deracemisation and enantioconvergent transformations. Moreover, a much greater understanding is being developed of the parameters that can affect the stereochemical outcome of the reaction (e.g. solvent, substrate design, immobilization and directed evolution).

Catalysis↗
PubMed1998

Combinatorial protein design by in vitro recombination.

DNA recombination is a powerful engine for the creation of new phenotypes. Recently, methods for in vitro DNA recombination (DNA shuffling) have been developed and applied to the evolution of novel molecules in the laboratory. An exciting new development is the shuffling of homologous genes to create diversity for directed evolution.

DNA↗
PubMed1997

Test of synergistic interactions among deleterious mutations in bacteria.

Identifying the forces responsible for the origin and maintenance of sexuality remains one of the greatest unsolved problems in biology. The mutational deterministic hypothesis postulates that sex is an adaptation that allows deleterious mutations to be purged from the genome; it requires synergistic interactions, which means that two mutations would be more harmful together than expected from their separate effects. We generated 225 genotypes of Escherichia coli carrying one, two or three successive mutations and measured their fitness relative to an unmutated competitor. The relationship between mutation number and average fitness is nearly log-linear. We also constructed 27 recombinant genotypes having pairs of mutations whose separate and combined effects on fitness were determined. Several pairs exhibit significant interactions for fitness, but they are antagonistic as often as they are synergistic. These results do not support the mutational deterministic hypothesis for the evolution of sex.

Directed Molecular Evolution↗
PubMed2005

[Directed evolution of D-lactonohydrolase by error prone PCR and DNA shuffling].

D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.

Carboxylic Ester Hydrolases↗
PubMed2003

Increased rigidity of domain structures enhances the stability of a mutant enzyme created by directed evolution.

A mutant of kanamycin nucleotidyltransferase (KNT) was previously created by directed evolution. This mutant, HTK, has 19 amino acid substitutions, which increase the thermostability by 20 degrees C. In this study, we have examined to what extent each mutation contributes to the increased stability and analyzed how the mutations affect the structure of KNT at 72 degrees C using molecular dynamics simulations. The effects of some mutations on the stability are simply additive, but those of others are cooperative. Mutations with large effects on the stability are introduced into the N-terminal domain, which appears to be less stable than the C-terminal domain. Results of the molecular dynamics simulations have indicated that the rigidity of the domain structures is increased by the mutations: at 72 degrees C, the intradomain fluctuations of HTK are decreased, and in turn, its interdomain motions are pronounced, whereas the structure of the preevolved KNT fluctuates randomly. Chemical modification experiments of cysteine residues have shown that the cysteine residues of HTK are less accessible to an SH reagent than those of the preevolved KNT. The present results suggest that the 19 mutations of HTK stabilize KNT by affecting the dynamic behavior of the structure of this enzyme without significantly changing its static overall structure.

Alanine↗
PubMed2001

Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design.

Do non-active-site residues participate in protein function in a more direct way than just by holding the static framework of the protein molecule? If so, how important are they? As a model to answer these questions, ATB17, which is a mutant of aspartate aminotransferase created by directed evolution, is an ideal system because it shows a 10(6)-fold increase in the catalytic efficiency for valine but most of its 17 mutated residues are non-active-site residues. To analyze the roles of the mutations in the altered function, we divided the mutations into four groups, namely, three clusters and the remainder, based on their locations in the three-dimensional structure. Mutants with various combinations of the clusters were constructed and analyzed, and the data were interpreted in the context of the structure-function relationship of this enzyme. Each cluster shows characteristic effects: for example, one cluster appears to enhance the catalytic efficiency by fixing the conformation of the enzyme to that of the substrate-bound form. The effects of the clusters are largely additive and independent of each other. The present results illustrate how a protein function is dramatically modified by the accumulation of many seemingly inert mutations of non-active-site residues.

Amino Acid Substitution↗
PubMed2005

Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase.

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli beta-galactosidase (BGAL) variants with enhanced beta-fucosidase activity (tenfold increase in k(cat)/K(M) in reactions with the novel para-nitrophenyl-beta-d-fucopyranoside substrate; 39-fold decrease in reactivity with the "native"para-nitrophenyl-beta-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of beta-fucosidases that are significantly more active (180-fold increase in k(cat)/K(M) in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.

Base Sequence↗
PubMedDate not supplied

Pooling for improved screening of combinatorial libraries for directed evolution.

Following diversity generation in combinatorial protein engineering, a significant amount of effort is expended in screening the library for improved variants. Pooling, or combining multiple cells into the same assay well when screening, is a means to increase throughput and screen a larger portion of the library with less time and effort. We have developed and validated a Monte Carlo simulation model of pooling and used it to screen a library of beta-galactosidase mutants randomized in the active site to increase their activity toward fucosides. Here, we show that our model can successfully predict the number of highly improved mutants obtained via pooling and that pooling does increase the number of good mutants obtained. In unpooled conditions, we found a total of three mutants with higher activity toward p-nitrophenyl-beta-D-fucoside than that of the wild-type beta-galactosidase, whereas when pooling 10 cells per well we found a total of approximately 10 improved mutants. In addition, the number of "supermutants", those with the highest activity increase, was also higher when pooling was used. Pooling is a useful tool for increasing the efficiency of screening combinatorial protein engineering libraries.

Binding Sites↗
PubMed2005

Directed evolution of copy number of a broad host range plasmid for metabolic engineering.

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.

Directed Molecular Evolution↗
PubMed2006

Directed evolution of plant basic helix-loop-helix transcription factors for the improvement of transactivational properties.

Myc-RP from Perilla frutescens and Delila from Antirrhinum majus, two plant basic helix-loop-helix transcription factors (bHLH TFs) involved in the flavonoid biosynthetic pathway, have been used for the improvement of transactivational properties by directed evolution. Through two rounds of DNA shuffling, Myc-RP variants with up to 70-fold increase in transcriptional activities have been identified using a yeast transactivation system. In a tobacco protoplast transient expression assay, one of the most improved variants, M2-1, also shows significant increase of transactivation. The majority of resulting mutations (approximately 53%) are localized in the acidic (activation) domains of the improved Myc-RP variants. In variant M2-1, three of the four mutations (L301P/N354D/S401F) are in the acidic domain. The fourth mutation (K157M) is localized to a helix within the N-terminal interaction domain. Combinatorial site-directed mutagenesis reveals that, while the acidic domain mutations contribute modestly to the increase in activity, the K157M substitution is responsible for 80% of the improvement observed in variant M2-1. The transactivation activity of the K157M/N354D double mutant is equal to that of M2-1. These results suggest that the interaction domain plays a critical role in transactivation of these bHLH TFs. Delila variants have also been screened for increased activities toward the Arabidopsis chalcone synthase (CHS) promoter, a pathway promoter that responds weakly to the bHLH TFs. Variants with increased activity on the CHS promoter, while maintaining wildtype-level activities on the naturally responsive dihydroflavonol reductase promoter, have been obtained. This study demonstrates that functional properties of TFs can be modified by directed evolution.

Acyltransferases↗
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↗