Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

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Cd-Specific Mutants of Mercury-Sensing Regulatory Protein MerR, Generated by Directed Evolution

Applied and Environmental Microbiology ◽

10.1128/aem.00662-11 ◽

2011 ◽

Vol 77 (17) ◽

pp. 6215-6224 ◽

Cited By ~ 18

Author(s):

Kaisa M. Hakkila ◽

Pia A. Nikander ◽

Sini M. Junttila ◽

Urpo J. Lamminmäki ◽

Marko P. Virta

Keyword(s):

Amino Acid ◽

Directed Evolution ◽

Metal Binding ◽

Fluorescent Protein ◽

Regulatory Protein ◽

Screening Strategy ◽

Mercury Resistance ◽

Content Type ◽

Green Fluorescent ◽

Mercury Sensing

ABSTRACTThe mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via themeroperator/promoter. AnEscherichia colicell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.

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Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors

Blood ◽

10.1182/blood.v112.11.3532.3532 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3532-3532

Author(s):

Stephan Maersch ◽

Anke Huber ◽

Michael Hallek ◽

Hildegard Buening ◽

Luca Perabo

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gene Transfer ◽

Directed Evolution ◽

Neutralizing Antibodies ◽

Rational Design ◽

Multiple Point ◽

Repeated Treatment ◽

Therapeutic Gene ◽

Adeno Associated Virus

Abstract Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.

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Creation of an Amino Acid Network of Structurally Coupled Residues in the Directed Evolution of a Thermostable Enzyme

Angewandte Chemie ◽

10.1002/ange.200904209 ◽

2009 ◽

Vol 121 (44) ◽

pp. 8418-8422 ◽

Cited By ~ 13

Author(s):

Manfred T. Reetz ◽

Pankaj Soni ◽

Juan Pablo Acevedo ◽

Joaquin Sanchis

Keyword(s):

Amino Acid ◽

Directed Evolution ◽

Thermostable Enzyme

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Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

Canadian Journal of Microbiology ◽

10.1139/w01-118 ◽

2001 ◽

Vol 47 (12) ◽

pp. 1088-1094 ◽

Cited By ~ 21

Author(s):

Yew-Loom Chen ◽

Tsung-Yin Tang ◽

Kuo-Joan Cheng

Keyword(s):

Amino Acid ◽

Directed Evolution ◽

Catalytic Domain ◽

Specific Activity ◽

Synergistic Effects ◽

High Specific Activity ◽

Site Directed Mutagenesis ◽

Amino Acid Substitutions ◽

Wild Type ◽

Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min1·(mg protein)1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

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