High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2

Abécassis, Valérie; Pompon, Denis; Truan, Gilles

doi:10.1093/nar/28.20.e88

Cited by 105 publications

(88 citation statements)

References 47 publications

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“…In vitro evolution. Gene libraries of KE07 were created by random mutagenesis using error-prone PCR with 'wobble' base analogues dPTP and 8-oxo-dGTP 26 using the Genemorph PCR mutagenesis kit (Stratagene), and by DNA shuffling of the most active variants 27 . In certain rounds, shuffling included the spiking of synthetic oligonucleotides that expanded the diversity at selected residues 28 .…”

Section: Methods Summarymentioning

confidence: 99%

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Kemp elimination catalysts by computational enzyme design

Röthlisberger¹,

Khersonsky²,

Wollacott³

et al. 2008

Nature

1,184

1,237

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The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10 5 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a .200-fold increase in k cat /K m (k cat /K m of 2,600 M 21 s 21 and k cat /k uncat of .10 6 ). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.Naturally occurring enzymes are extraordinarily efficient catalysts 1 . They bind their substrates in a well-defined active site with precisely aligned catalytic residues to form highly active and selective catalysts for a wide range of chemical reactions under mild conditions. Nevertheless, many important synthetic reactions lack a naturally occurring enzymatic counterpart. Hence, the design of stable enzymes with new catalytic activities is of great practical interest, with potential applications in biotechnology, biomedicine and industrial processes. Furthermore, the computational design of new enzymes provides a stringent test of our understanding of how naturally occurring enzymes work. In the past several years, there has been exciting progress in designing new biocatalysts 2,3 .Here we describe the use of our recently developed computational enzyme design methodology 4 to create new enzyme catalysts for a reaction for which no naturally occurring enzyme exists: the Kemp elimination 5,6 . The reaction, shown in Fig. 1a, has been extensively studied as an activated model system for understanding the catalysis of proton abstraction from carbon-a process that is normally restricted by high activation-energy barriers 7,8 . Computational design methodThe first step in our protocol for designing new enzymes is to choose a catalytic mechanism and then to use quantum mechanical transition state calculations to create an idealized active site with protein functional groups positioned so as to maximize transition state stabilization (Fig. 1b). The key step for the Kemp elimination is deprotonation of a carbon by a general base. We chose two different catalytic bases for this purpose: first, the carboxyl group of an aspartate or glutamate side chain, and, second, the imidazole of a histidine positioned and polarized by the carboxyl group of an aspartate or glutamate (we refer to this combination as a His-Asp dyad). The two choices have complementary strengths and weaknesses. The advantage of the carboxylate...

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Section: Methods Summarymentioning

confidence: 99%

“…Round 2. The 23 most active variants isolated in the first round of screening were subjected to DNA shuffling in the presence of the designed template (20%) 27 to yield second-generation libraries. The most active variants of round 2 had lysate activities up to 15-fold higher than that of the KE07.…”

mentioning

confidence: 99%

Kemp elimination catalysts by computational enzyme design

Röthlisberger¹,

Khersonsky²,

Wollacott³

et al. 2008

Nature

1,184

1,237

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“…The genetic diversity in KE59 genes was generated by: error-prone PCR with mutazyme (Genemorph PCR mutagenesis kit, Stratagene) (39), gene shuffling (40), and mutations incorporation by spiking oligonucleotides during the assembly of DNA fragments (26). After mutagenesis and/or shuffling, the KE59 genes were recloned into the original pET29b plasmid (16).…”

Section: Methodsmentioning

confidence: 99%

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Khersonsky

Kiss

Röthlisberger

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

214

221

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Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cat ∕K m values of ≤5 × 10 4 M −1 s −1 ). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cat ∕K m , and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to >2,000-fold increase in catalytic efficiency, mainly via higher k cat values. The best KE59 variants exhibited k cat ∕K m values up to 0.6 × 10 6 M −1 s −1 , and k cat ∕k uncat values of ≤10 7 almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2-7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig. 1A). Activation of a carboxylate base catalyst by desolvation is efficiently mimicked by aprotic dipolar solvents such as acetonitrile and by various enzyme mimics. Effective alignment of the substrate and charge-dispersing interactions that stabilize the negatively charged transition state (TS) have also been achieved by various enzyme mimics that catalyze the Kemp elimination (8, 9). However, generation of a (wo)manmade active site that exhibits all these features and performs as well as natural enzymes, remains a challenge.Computational methods for predicting structure from sequence at atomic accuracy provide a new approach to enzyme engineering (10-12). The design involves two steps: (i) designing an active-site configuration that may confer efficient catalysis, (ii) computing a sequence that confers the desired configuration. Both steps are currently far from optimal, as the catalytic efficiency of designed enzymes falls far behind that of natural enzymes. Further, as with other enzyme mimics, computational designs tackle activated model systems and fail to catalyze cha...

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“…These techniques are attractive for their simplicity, as they require only a straightforward transformation step. In vivo homologous recombination has also been used extensively in the context of library generation for directed evolution applications; the highly efficient recombination machinery of organisms such as Saccharomyces cerevisiae has frequently been employed to build single-gene libraries containing 10 4 -10 10 variants (22)(23)(24)(25)(26). However, previously reported in vivo assembly techniques for multigene constructs have been low yielding, generating only tens to hundreds of recombinants at a time and making them impractical for the construction of libraries.…”

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confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

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The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae , and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10 4 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

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High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2

Cited by 105 publications

References 47 publications

Kemp elimination catalysts by computational enzyme design

Kemp elimination catalysts by computational enzyme design

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

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