New Concepts for Increasing the Efficiency in Directed Evolution of Stereoselective Enzymes

Sun, Zhoutong; Wikmark, Ylva; Bäckvall, Jan‐E.; Reetz, Manfred T.

doi:10.1002/chem.201504406

Cited by 81 publications

(58 citation statements)

References 67 publications

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“…To test strategy 2 (Scheme ), only library C was considered for S ‐selective library design (Table ). Accordingly, the amino‐acid alphabets VW, VLA, GSL, and NHQ were chosen for residues A85, I86, W110, and L294, respectively; these codon decisions were made on the basis of the exploratory NNK experiments (Table S1) .…”

Section: Resultssupporting

confidence: 53%

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Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Lonsdale

Yao

et al. 2018

ChemBioChem

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Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets. By using the synthetically difficult-to-reduce prochiral ketone tetrahydrofuran-3-one as a substrate, highly R- and S-selective variants were obtained (92-99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi-Dunitz trajectory.

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Section: Resultssupporting

confidence: 53%

“…This approach was shown to be successful in the previous TbSADH study . However, it is also possible to use a different codon degeneracy at each individual residue in a single SM experiment (dubbed “strategy 2”; Scheme ) . This option requires more structural and/or computational data.…”

Section: Introductionmentioning

confidence: 99%

Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Lonsdale

Yao

et al. 2018

ChemBioChem

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“…It also needs to be pointed out that two ways for applying SM are possible: (1) Use of one and the same codon degeneracy for randomizing combinatorially the entire multi-residue site; or (2) Use of a different codon degeneracy at each individual position of a multi-residue site. Both types of SM are performed in a single experiment (Sun et al 2016a).…”

Section: Introductionmentioning

confidence: 99%

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

Sun

Salas

Siirola

et al. 2016

Bioresour. Bioprocess.

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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.

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“…This has significantly reduced the cost of development and subsequent production of enzymes at industrial scale. -Advances in protein engineering, using directed evolution techniques [17,18], have enabled optimization of enzyme performance under the challenging conditions encountered in industrial-scale processes, namely, high (stereo)selectivities, activities and space-time yields with non-natural substrates at high substrate concentrations, in the presence of organic solvents.In short, more enzymes are available, the enzymes are better, and, thanks to recombinant DNA technology, they can be economically produced on an industrial scale, thus enabling the design of improved industrial biocatalysts [19]. Notwithstanding these numerous benefits of using enzymes, their industrial application is often hampered by a lack of long-term stability and difficult recovery and re-use.…”

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confidence: 99%

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CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

2019

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Biocatalysis has emerged in the last decade as a pre-eminent technology for enabling the envisaged transition to a more sustainable bio-based economy. For industrial viability it is essential that enzymes can be readily recovered and recycled by immobilization as solid, recyclable catalysts. One method to achieve this is via carrier-free immobilization as cross-linked enzyme aggregates (CLEAs). This methodology proved to be very effective with a broad selection of enzymes, in particular carbohydrate-converting enzymes. Methods for optimizing CLEA preparations by, for example, adding proteic feeders to promote cross-linking, and strategies for making the pores accessible for macromolecular substrates are critically reviewed and compared. Co-immobilization of two or more enzymes in combi-CLEAs enables the cost-effective use of multiple enzymes in biocatalytic cascade processes and the use of "smart" magnetic CLEAs to separate the immobilized enzyme from other solids has raised the CLEA technology to a new level of industrial and environmental relevance. Magnetic-CLEAs of polysaccharide-converting enzymes, for example, are eminently suitable for use in the conversion of first and second generation biomass.Catalysts 2019, 9, 261 2 of 31 and deprotection steps. This affords synthetic routes that, compared with conventional organic syntheses, are more step economic, more energy efficient, generate less waste and provide products in exquisite stereochemical purities that are difficult to compete with.Consequently, in the last two decades biocatalysis has emerged as an important technology for meeting the growing demand for green and sustainable processing [2][3][4]. This embraces the whole gamut of chemicals manufacture, from the enantioselective synthesis of chiral drugs [5][6][7][8][9][10][11][12] to the conversion of renewable biomass to liquid fuels and commodity chemicals [13][14][15]. This raises the perennial question: if biocatalysis is so superior why has it not been extensively used in the past? The answer is simple: thanks to advances in molecular biology and biotechnology, biocatalysis has undergone a spectacular leap forward in the last two decades:-Many more enzymes have been identified through (meta)genome mining, that is the in silico analysis of publicly accessible genome sequence data bases that have been generated as a result of next generation genome sequencing [16]. -Advances in gene synthesis have enabled the synthesis of identified genes, ready for cloning into a host production organism, in a few weeks at relatively low cost. This has significantly reduced the cost of development and subsequent production of enzymes at industrial scale. -Advances in protein engineering, using directed evolution techniques [17,18], have enabled optimization of enzyme performance under the challenging conditions encountered in industrial-scale processes, namely, high (stereo)selectivities, activities and space-time yields with non-natural substrates at high substrate concentrations, in the presence of organic solve...

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New Concepts for Increasing the Efficiency in Directed Evolution of Stereoselective Enzymes

Cited by 81 publications

References 67 publications

Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

Methodology Development in Directed Evolution: Exploring Options when Applying Triple‐Code Saturation Mutagenesis

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

CLEAs, Combi-CLEAs and ‘Smart’ Magnetic CLEAs: Biocatalysis in a Bio-Based Economy

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