Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolution

Jones, D. Dafydd; Arpino, James A. J.; Baldwin, Amy; Edmundson, Matthew Charles

doi:10.1007/978-1-4939-1053-3_11

Cited by 11 publications

(6 citation statements)

References 20 publications

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“…S5). Introduction of randomised larger insertions (+12 bp and beyond) is achievable 52 , but would lead to libraries that are larger than the typical screening capacity. Finally, the procedure is technically straightforward, consisting of transposition and cloning steps, and does not require access to specialized DNA synthesis equipment (as in ref.…”

Section: Discussionmentioning

confidence: 99%

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

et al. 2020

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Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposonbased mutagenesis approach (TRIAD) to generate libraries of random variants with short inframe InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20-to 35-fold increased k cat /K M in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in k cat rather than just in K M , suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.

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Section: Discussionmentioning

confidence: 99%

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

et al. 2020

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“…580–583 However, a common issue with site-directed mutagenesis methods is the large number of steps involved and the limited number of positions that can be efficiently targeted at a time. The ability to mutate residues in multiple positions in a sequence is of particular interest as this can be used to address the question of combinatorial mutations simultaneously.…”

Section: Diversity Creation and Library Designmentioning

confidence: 99%

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

et al. 2015

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“…We used TRIAD 12 mutagenesis to generate single amino acid insertion and deletion libraries of a stabilized bi-functional ancestral enzyme 15 of haloalkane dehalogenase and Renilla-luciferase enzymes (Figure 1). However, other published methods for introducing insertions and deletions could be used [23][24][25][26] . The most potent insertion variant, AncINS, has both an insertion and a substitution in the α4 helix.…”

Section: Discussionmentioning

confidence: 99%

Engineering Protein Dynamics of Ancestral Luciferase

Schenkmayerova¹,

Pinto²,

Toul³

et al. 2020

Preprint

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Insertion-deletion mutations are sources of major functional innovations in naturally evolved proteins, but directed evolution methods rely primarily on substitutions. Here, we report a powerful strategy for engineering backbone dynamics based on InDel mutagenesis of a stable and evolvable template, and its validation in application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive multidisciplinary analysis linked the conformational flexibility of a loop-helix fragment to binding of the bulky substrate coelenterazine. The fragment’s key role in extant Renilla luciferase was confirmed by transplanting it into the ancestor. This increased its catalytic efficiency 7,000-fold, and fragment-containing mutants showed highly stable glow-type bioluminescence with 100-fold longer half-lives than the flash-type Renilla luciferase RLuc8, thereby addressing a limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a robust template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of a dynamic feature, provides a potent strategy for discovering protein modifications with globally disruptive but functionally innovative effects.

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Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolution

Cited by 11 publications

References 20 publications

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

Engineering Protein Dynamics of Ancestral Luciferase

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