Directed Evolution and Rational Design of Mechanosensitive Channel MscCG2 for Improved Glutamate Excretion Efficiency

Genetic code expansion depends on the directed evolution of aaRS to recognize non-canonical amino acids. Herein, we reported a function-based method that enables rapidly evolving aaRS for acylated lysine derivatives.

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Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

Microorganisms ◽

10.3390/microorganisms8040519 ◽

2020 ◽

Vol 8 (4) ◽

pp. 519

Author(s):

Lisheng Xu ◽

Fangkai Han ◽

Zeng Dong ◽

Zhaojun Wei

Keyword(s):

Thermal Stability ◽

Amino Acid ◽

Directed Evolution ◽

Amino Acid Residue ◽

Rational Design ◽

Molecular Engineering ◽

Mutant Enzyme ◽

Tryptophan Synthase ◽

Mutant Library ◽

Thermal Stability Of

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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Improving the thermostability of a GH11 xylanase by directed evolution and rational design guided by B-factor analysis

Enzyme and Microbial Technology ◽

10.1016/j.enzmictec.2020.109720 ◽

2021 ◽

Vol 143 ◽

pp. 109720

Author(s):

Hongguan Xing ◽

Gen Zou ◽

Chunyan Liu ◽

Shunxing Chai ◽

Xing Yan ◽

...

Keyword(s):

Factor Analysis ◽

Directed Evolution ◽

Rational Design

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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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Directed evolution combined with rational design increases activity of GpdQ toward a non-physiological substrate and alters the oligomeric structure of the enzyme

Protein Engineering Design and Selection ◽

10.1093/protein/gzr048 ◽

2011 ◽

Vol 24 (12) ◽

pp. 861-872 ◽

Cited By ~ 14

Author(s):

Sylvia H-C. Yip ◽

Jee-Loon Foo ◽

Gerhard Schenk ◽

Lawrence R. Gahan ◽

Paul D. Carr ◽

...

Keyword(s):

Directed Evolution ◽

Rational Design ◽

Oligomeric Structure

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65. Rational Design Meets Directed Evolution for Tailoring the AAV Capsid

Molecular Therapy ◽

10.1016/s1525-0016(16)37506-2 ◽

2010 ◽

Vol 18 ◽

pp. S26

Keyword(s):

Directed Evolution ◽

Rational Design

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A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

Biological Chemistry ◽

10.1515/bc.2007.153 ◽

2007 ◽

Vol 388 (12) ◽

pp. 1291-1300 ◽

Cited By ~ 10

Author(s):

Ai-Sheng Xiong ◽

Ri-He Peng ◽

Jing Zhuang ◽

Jin-Ge Liu ◽

Feng Gao ◽

...

Keyword(s):

Directed Evolution ◽

Dna Sequences ◽

Active Sites ◽

Rational Design ◽

Specific Activity ◽

Dna Shuffling ◽

Gene Encoding ◽

Library Construction ◽

Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

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Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

10.1101/2020.09.27.315697 ◽

2020 ◽

Author(s):

Shuqin Zhou ◽

Siyu Feng ◽

David Brown ◽

Bo Huang

Keyword(s):

Directed Evolution ◽

Signal Amplification ◽

Mammalian Cells ◽

Rational Design ◽

Fluorescent Proteins ◽

Protein Labeling ◽

Small Fragment ◽

Wide Range ◽

Split System ◽

Low Expression

AbstractThe flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.

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Directed Evolution of Protein Catalysts

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-062917-012034 ◽

2018 ◽

Vol 87 (1) ◽

pp. 131-157 ◽

Cited By ~ 135

Author(s):

Cathleen Zeymer ◽

Donald Hilvert

Keyword(s):

Directed Evolution ◽

Rational Design ◽

Chemical Transformations ◽

Catalytic Promiscuity ◽

Catalytic Activities ◽

Effective Strategies ◽

Wide Range ◽

Protein Properties ◽

New Protein ◽

Modify Protein

Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of biocatalytic applications. Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are applied to modify protein properties, enhance catalytic activities, or develop completely new protein catalysts for non-natural chemical transformations. This review briefly surveys the experimental methods used to generate genetic diversity and screen or select for improved enzyme variants. Emphasis is placed on a key challenge, namely how to generate novel catalytic activities that expand the scope of natural reactions. Two particularly effective strategies, exploiting catalytic promiscuity and rational design, are illustrated by representative examples of successfully evolved enzymes. Opportunities for extending these approaches to more complex biocatalytic systems are also considered.

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