Enzyme engineering in biomimetic compartments

Colin, Pierre-Yves; Zinchenko, Anastasia; Hollfelder, Florian

doi:10.1016/j.sbi.2015.06.001

Cited by 59 publications

(50 citation statements)

References 81 publications

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“…To produce the protein, the HaeIII methyltransferase, they used cell-free protein synthesis (see above). [30] This technique alleviates the step of ligating the gene into a plasmid and using the resulting library for the transformation of an organism (Fig. 2d).…”

Section: Applications In Vitromentioning

confidence: 99%

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Compartmentalization – A Prerequisite for Maintaining and Changing an Identity

Rottmann

Ward

Panke³

2016

Chimia

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The chemical manipulation of DNA is much more convenient than the manipulation of the bioproducts, such as enzymes, that it encodes. The optimization of bioproducts requires cycles of diversification of DNA followed by read-out of the information into the bioproduct. Maintaining the link between the information - the genotype - and the properties of the bioproduct - the phenotype - through some form of compartmentalization is therefore an essential aspect in directed evolution. While the ideal compartment is a biological cell, many projects involving more radical changes in the bioproduct, such as the introduction of novel cofactors, may not be suitable for expression of the information in cells, and alternative in vitro methods have to be applied. Consequently, the possibility to produce simple and advanced micro compartments at high rates and to combine them with the ability to translate the information into proteins represents a unique opportunity to explore demanding enzyme engineering projects that require the evaluation of at least hundreds of thousands of enzyme variants over multiple generations.

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Section: Applications In Vitromentioning

confidence: 99%

“…Simple representation of expression systems that can be used in combination with compartmentalization techniques. [30] a) Expression of a specific gene product in a cell and subsequent lysis after encapsulation. b) Secretion of the gene product after encapsulation of the host organism.…”

Section: Applications In Vivo Cell Lysismentioning

confidence: 99%

Compartmentalization – A Prerequisite for Maintaining and Changing an Identity

Rottmann

Ward

Panke³

2016

Chimia

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“…Although this approach relies on screening rather than selection, the automated, high-throughput nature of FACS allows sorting of up to 10 7 compartments per hour 25 , and hence is a powerful alternative to selection systems. However, as flow cytometry requires aqueous media, the compartments used for selection had to be emulsified twice to obtain water-oil-water double emulsions, which are highly polydisperse 26 . The high variability between ‘double-bagged’ compartments makes it difficult to precisely discriminate incremental improvements in enzyme activities and reduces enrichment efficiency.…”

Section: Introductionmentioning

confidence: 99%

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

et al. 2017

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Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a ‘partner’ function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take ~3–5 d, whereas a whole directed evolution can be performed in 3–10 rounds, depending on selection efficiency.

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“…At systems level, the implementation of biomimesis allows for testing the hierarchy of importance of the various structure-function relationships in the natural systems. In the process of understanding the molecular and cellular aspects of living organisms, numerous examples of artificial enzymes [41][42][43][44], synthetic de novo designs of proteins [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59], artificial photo-driven proton pumps [60,61], and artificial cells with designated functionalities [62][63][64][65] have advanced the fields of biology, biochemistry and biophysics and provided unprecedented paradigms for bioengineering. At an organism-size level, examples for walking machines, which do not utilize wheels or roller, may seem somewhat impractical for the "optimal practical usage" of the current state of robotics.…”

Section: Introductionmentioning

confidence: 99%

Bioinspired approach toward molecular electrets: synthetic proteome for materials

Espinoza

Larsen-Clinton

Krzeszewski

et al. 2017

Pure and Applied Chemistry

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Molecular-level control of charge transfer (CT) is essential for both, organic electronics and solarenergy conversion, as well as for a wide range of biological processes. This article provides an overview of the utility of local electric fields originating from molecular dipoles for directing CT processes. Systems with ordered dipoles, i.e. molecular electrets, are the centerpiece of the discussion. The conceptual evolution from biomimicry to biomimesis, and then to biological inspiration, paves the roads leading from testing the understanding of how natural living systems function to implementing these lessons into optimal paradigms for specific applications. This progression of the evolving structure-function relationships allows for the development of bioinspired electrets composed of non-native aromatic amino acids. A set of such non-native residues that are electron-rich can be viewed as a synthetic proteome for hole-transfer electrets. Detailed considerations of the electronic structure of an individual residue prove of key importance for designating the points for optimal injection of holes (i.e. extraction of electrons) in electret oligomers. This multifaceted bioinspired approach for the design of CT molecular systems provides unexplored paradigms for electronic and energy science and engineering.

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Enzyme engineering in biomimetic compartments

Cited by 59 publications

References 81 publications

Compartmentalization – A Prerequisite for Maintaining and Changing an Identity

Compartmentalization – A Prerequisite for Maintaining and Changing an Identity

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

Bioinspired approach toward molecular electrets: synthetic proteome for materials

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