Exploring productive sequence space in directed evolution using binary patterning versus conventional mutagenesis strategies

Abstract

Background: Recent methodology development in directed evolution of stereoselective enzymes has shown that various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation of small and smart mutant libraries requiring minimal screening. Methods:In this endeavor, limonene epoxide hydrolase (LEH) has served as an experimental platform, the hydrolytic desymmetrization of cyclohexene oxide being the model reaction with formation of (R,R)-and (S,S)-cyclohexane-1,2-diol. This system has now been employed for testing reduced amino acid alphabets based on the Hecht concept of binary patterning, with and without additional hydrophobic amino acids. Results and Conclusions:It turns out that in binary pattern based saturation mutagenesis as applied to LEH, polar amino acids are seldom introduced. When applying binary patterning in combination with additional hydrophobic amino acids as building blocks in iterative saturation mutagenesis, excellent LEH variants were evolved for the production of both (R,R)-and (S,S)-diols (80-97 % ee), but again the introduction of polar amino acids occurs rarely. Docking computations explain the source of enhanced and inverted stereoselectivity. Some of the best variants are also excellent catalysts in the hydrolytic desymmetrization of other meso-epoxides, although both enantiomeric diols are not always accessible.

Simultaneous screening of multiple substrates with an unspecific peroxygenase enabled modified alkane and alkene oxyfunctionalisations

“…21,22 Another approach is to limit the available amino acid alphabet to solely include functionally relevant residues. 23,24 These extremely successful developments vastly increased the applicability of directed evolution.…”

Reductive Amination of Biobased Levulinic Acid to Unnatural Chiral γ-Amino Acid Using an Engineered Amine Dehydrogenase

“…With the advances of directed evolution, biocatalysis is increasingly applied as an environmentally friendly and sustainably alternative technology in the large-scale production of chiral chemicals. − Over time, numerous enzymatic routes catalyzed by lipases, amidases, acylases, nitrilases, hydantoinases, transaminases, ammonia lyases, aldolases, and amino acid dehydrogenases have been developed to provide access to chiral amino acids. Especially, numerous amino acid dehydrogenases , and several engineered amine dehydrogenases − created by evolution of amino acid dehydrogenases have been developed.…”

Improvement of the Activity and Stability of Starch-Debranching Pullulanase from Bacillus naganoensis via Tailoring of the Active Sites Lining the Catalytic Pocket

“…3−10 The combinatorial active-site-saturation test and iterative SM have been developed to create mutant libraries with reduced amino acid alphabets. 3,5,7,8,11 In addition, Sun et al developed a structure-guided triple-code-saturation-mutagenesis (TCSM) method for efficiently improving the stereoselectivity of limonene epoxide hydrolase and the enantioselectivity of alcohol dehydrogenase. 9,10 This method involved introduction of three rationally chosen amino acids as building blocks for SM at the randomization sites lining the catalytic and binding pocket of pullulanase.…”

“…The most commonly used methods for directed evolution are epPCR, DNA shuffling, and saturation mutagenesis (SM). Instead of iterative cycles of random amino-acid changes in a protein, more attention is focused on improving the efficiency of directed evolution by creating “small-and-smart” libraries. − The combinatorial active-site-saturation test and iterative SM have been developed to create mutant libraries with reduced amino acid alphabets. ,,,, In addition, Sun et al. developed a structure-guided triple-code-saturation-mutagenesis (TCSM) method for efficiently improving the stereoselectivity of limonene epoxide hydrolase and the enantioselectivity of alcohol dehydrogenase. , This method involved introduction of three rationally chosen amino acids as building blocks for SM at the randomization sites lining the catalytic and binding pocket of pullulanase.…”