Manipulating the Stereoselectivity of Limonene Epoxide Hydrolase by Directed Evolution Based on Iterative Saturation Mutagenesis

2010 ◽  
Vol 132 (44) ◽  
pp. 15744-15751 ◽  
Author(s):  
Huabao Zheng ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Epoxide Hydrolase ◽  
Saturation Mutagenesis ◽  
Iterative Saturation Mutagenesis

Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis

2009 ◽  
Vol 351 (18) ◽  
pp. 3287-3305 ◽  
Author(s):  
Despina J. Bougioukou ◽  
Sabrina Kille ◽  
Andreas Taglieber ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Enoate Reductase ◽  
Iterative Saturation Mutagenesis

Directed Evolution by Using Iterative Saturation Mutagenesis Based on Multiresidue Sites

ChemBioChem ◽  
2013 ◽  
Vol 14 (17) ◽  
pp. 2301-2309 ◽  
Author(s):  
Loreto P. Parra ◽  
Rubén Agudo ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Iterative Saturation Mutagenesis

Directed evolution of enantioselective enzymes: An unceasing catalyst source for organic chemistry

2010 ◽  
Vol 82 (8) ◽  
pp. 1575-1584 ◽  
Author(s):  
Manfred T. Reetz ◽  
Sheng Wu ◽  
Huabao Zheng ◽  
Shreenath Prasad
Keyword(s):  
Organic Chemistry ◽  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Powerful Method ◽  
Laboratory Evolution ◽  
Synthetic Organic Chemistry ◽  
Iterative Saturation Mutagenesis

Directed evolution has emerged as a powerful method for engineering essentially any catalytic parameter of enzymes for application in synthetic organic chemistry and biotechnology, including thermostability, substrate scope, and enantioselectivity. Enantioselectivity is especially crucial when applying biocatalysts to synthetic organic chemistry. This contribution focuses on recent methodology developments in laboratory evolution of stereoselective enzymes, hydrolases, and monooxygenases serving as the enzymes. Specifically, iterative saturation mutagenesis (ISM) has been developed as an unusually effective method to evolve enhanced or reversed enantioselectivity, broader substrate scope, and/or higher thermostability of enzymes.

scholarly journals Refactoring the Genetic Code for Increased Evolvability

2017 ◽  
Author(s):  
Gur Pines ◽  
James D. Winkler ◽  
Assaf Pines ◽  
Ryan T. Gill
Keyword(s):  
Genetic Code ◽  
Directed Evolution ◽  
Single Gene ◽  
Point Mutations ◽  
Saturation Mutagenesis ◽  
Mutagenic Potential ◽  
Single Nucleotide ◽  
Common Error ◽  
Mutational Landscape ◽  
Genetic Codes

AbstractThe standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.Significance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

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