Coupling genetic code expansion and metabolic engineering for synthetic cells

Völler, Jan‐Stefan; Budiša, Nediljko

doi:10.1016/j.copbio.2017.02.002

Cited by 58 publications

(39 citation statements)

References 40 publications

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“…A poor protein loading efficiency due to immobilization only to surface might be overcome by using highly porous materials with a very large surface area. Another issue of a relatively high cost of bioorthogonal non‐natural amino acids might be addressed by combining genetic code expansion and metabolic engineering . An example of bioorthogonal non‐natural amino acid metabolic engineering was reported, where azidohomoalanine was produced from cells using inorganic azide.…”

Section: Resultsmentioning

confidence: 99%

Purification‐Free, Target‐Selective Immobilization of a Protein from Cell Lysates

Cha

Kwon

2018

Biotechnology Journal

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Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization).

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Section: Resultsmentioning

confidence: 99%

Purification‐Free, Target‐Selective Immobilization of a Protein from Cell Lysates

Cha

Kwon

2018

Biotechnology Journal

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“…In the most common approach, amino acid uptake is artificially controlled by feeding auxotrophs with NCAAs [33,34,35] (Figure 2a). Attempts to control NCAA synthesis have involved supplying organisms with NCAA precursors [57,58,59,60,61,62] (Figure 2b), which is also known as metabolic engineering [63,64]. In one example, the precursor l -β-thieno [3,2-b]pyrrolyl ([3,2]Trp) was fed to a tryptophan (Trp)-auxotrophic E. coli capable of synthesizing [3,2]Tpa (a Trp analog) to generate mutants that could propagate on l -β-(thieno [3,2-b]pyrrolyl)alanine ([3,2]Tpa) [57] (Figure 2b).…”

Section: Genetic Code Engineeringmentioning

confidence: 99%

Efforts and Challenges in Engineering the Genetic Code

Lin

Chan

2017

Life

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This year marks the 48th anniversary of Francis Crick’s seminal work on the origin of the genetic code, in which he first proposed the “frozen accident” hypothesis to describe evolutionary selection against changes to the genetic code that cause devastating global proteome modification. However, numerous efforts have demonstrated the viability of both natural and artificial genetic code variations. Recent advances in genetic engineering allow the creation of synthetic organisms that incorporate noncanonical, or even unnatural, amino acids into the proteome. Currently, successful genetic code engineering is mainly achieved by creating orthogonal aminoacyl-tRNA/synthetase pairs to repurpose stop and rare codons or to induce quadruplet codons. In this review, we summarize the current progress in genetic code engineering and discuss the challenges, current understanding, and future perspectives regarding genetic code modification.

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“…While SPI can be preferred for production of proteins with smaller and simpler amino acid substrates and analogous of hydrophobic amino acids, the SCS is generally preferred for aromatic or bulky non‐canonical substitutes. Only recently an orthogonal synthetase suitable for a small substrate, S ‐allyl‐cysteine was developed and general importance of metabolic engineering coupling with the expanded genetic code for the design of synthetic cells has been elaborated .…”

Section: Code Engineering and Synthetic Life – Does It Fit Together?mentioning

confidence: 99%

“…Only recently an orthogonal synthetase suitable for a small substrate, S-allyl-cysteine was developed [34] and general importance of metabolic engineering coupling with the expanded genetic code for the design of synthetic cells has been elaborated [35]. It has also been demonstrated in several studies that certain bacterial enzymes can be adjusted in such a way that a non-canonical amino acid incorporation can become vital for a microbial culture [36][37][38].…”

Section: Code Engineering and Synthetic Lifedoes It Fit Together?mentioning

confidence: 99%

Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

Kubyshkin

Budiša

2017

Biotechnology Journal

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The main goal of synthetic biology (SB) is the creation of biodiversity applicable for biotechnological needs, while xenobiology (XB) aims to expand the framework of natural chemistries with the non-natural building blocks in living cells to accomplish artificial biodiversity. Protein and proteome engineering, which overcome limitation of the canonical amino acid repertoire of 20 (+2) prescribed by the genetic code by using non-canonic amino acids (ncAAs), is one of the main focuses of XB research. Ideally, estranging the genetic code from its current form via systematic introduction of ncAAs should enable the development of bio-containment mechanisms in synthetic cells potentially endowing them with a "genetic firewall" i.e. orthogonality which prevents genetic information transfer to natural systems. Despite rapid progress over the past two decades, it is not yet possible to completely alienate an organism that would use and maintain different genetic code associations permanently. In order to engineer robust bio-contained life forms, the chemical logic behind the amino acid repertoire establishment should be considered. Starting from recent proposal of Hartman and Smith about the genetic code establishment in the RNA world, here the authors mapped possible biotechnological invasion points for engineering of bio-contained synthetic cells equipped with non-canonical functionalities.

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Coupling genetic code expansion and metabolic engineering for synthetic cells

Cited by 58 publications

References 40 publications

Purification‐Free, Target‐Selective Immobilization of a Protein from Cell Lysates

Purification‐Free, Target‐Selective Immobilization of a Protein from Cell Lysates

Efforts and Challenges in Engineering the Genetic Code

Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

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