Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Genetic Code Expansion of Vibrio natriegens

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.594429 ◽

2021 ◽

Vol 9 ◽

Author(s):

Eden Ozer ◽

Lital Alfonta

Keyword(s):

Genetic Code ◽

Unnatural Amino Acids ◽

Unnatural Amino Acid ◽

Negative Bacterium ◽

Amino Acid Incorporation ◽

Gram Negative ◽

E Coli ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

First Time

Escherichia coli has been considered as the most used model bacteria in the majority of studies for several decades. However, a new, faster chassis for synthetic biology is emerging in the form of the fast-growing gram-negative bacterium Vibrio natriegens. Different methodologies, well established in E. coli, are currently being adapted for V. natriegens in the hope to enable a much faster platform for general molecular biology studies. Amongst the vast technologies available for E. coli, genetic code expansion, the incorporation of unnatural amino acids into proteins, serves as a robust tool for protein engineering and biorthogonal modifications. Here we designed and adapted the genetic code expansion methodology for V. natriegens and demonstrate an unnatural amino acid incorporation into a protein for the first time in this organism.

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Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells

Molecular BioSystems ◽

10.1039/b904228h ◽

2009 ◽

Vol 5 (9) ◽

pp. 931 ◽

Cited By ~ 46

Author(s):

Jeffrey K. Takimoto ◽

Katrina L. Adams ◽

Zheng Xiang ◽

Lei Wang

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation

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Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1

Journal of the American Chemical Society ◽

10.1021/ja5069728 ◽

2014 ◽

Vol 136 (44) ◽

pp. 15577-15583 ◽

Cited By ~ 123

Author(s):

Wolfgang H. Schmied ◽

Simon J. Elsässer ◽

Chayasith Uttamapinant ◽

Jason W. Chin

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation

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High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Applied and Environmental Microbiology ◽

10.1128/aem.03417-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1718-1725 ◽

Cited By ~ 16

Author(s):

Masaomi Minaba ◽

Yusuke Kato

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Unnatural Amino Acids ◽

Mammalian Cells ◽

Expression System ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Translational Efficiency ◽

Site Specific ◽

Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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Evolving Bacterial Fitness with an Expanded Genetic Code

10.1101/169409 ◽

2017 ◽

Author(s):

Drew S. Tack ◽

Austin C. Cole ◽

R. Shroff ◽

B.R. Morrow ◽

Andrew D. Ellington

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acid ◽

Noncanonical Amino Acids ◽

Bacterial Fitness ◽

Genetic Code Expansion ◽

Encoding Strategy ◽

Translation Systems ◽

Expanded Genetic Code

AbstractEvolution has for the most part used the canonical 20 amino acids of the natural genetic code to construct proteins. While several theories regarding the evolution of the genetic code have been proposed, experimental exploration of these theories has largely been restricted to phylogenetic and computational modeling. The development of orthogonal translation systems has allowed noncanonical amino acids to be inserted at will into proteins. We have taken advantage of these advances to evolve bacteria to accommodate a 21 amino acid genetic code in which the amber codon ambiguously encodes either 3-nitro-L-tyrosine or stop. Such an ambiguous encoding strategy recapitulates numerous models for genetic code expansion, and we find that evolved lineages first accommodate the unnatural amino acid, and then begin to evolve on a neutral landscape where stop codons begin to appear within genes. The resultant lines represent transitional intermediates on the way to the fixation of a functional 21 amino acid code.

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Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

10.1101/742726 ◽

2019 ◽

Author(s):

Yonatan Chemla ◽

Eden Ozer ◽

Michael Shaferman ◽

Ben Zaad ◽

Rambabu Dandela ◽

...

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Unnatural Amino Acids ◽

High Efficiency ◽

Living Cells ◽

Trna Synthetase ◽

Complex Nature ◽

Reporter Protein ◽

A Cell ◽

Genetic Code Expansion

ABSTRACTGenetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

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Sterilization of drug-resistant influenza virus through genetic interference: use of unnatural amino acid-engineered virions

10.1101/2021.12.04.471209 ◽

2021 ◽

Author(s):

Xuesheng Wu ◽

Zhetao Zheng ◽

Hongmin Chen ◽

Haishuang Lin ◽

Yuelin Yang ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Influenza A ◽

Antiviral Agent ◽

Unnatural Amino Acid ◽

Drug Resistant ◽

Genetic Code Expansion

AbstractThe frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

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Optimization of Expanded Genetic Codes via Genetic Algorithms

10.5753/eniac.2018.4440 ◽

2018 ◽

Author(s):

Maísa de Carvalho Silva ◽

Lariza Laura De Oliveira ◽

Renato Tinós

Keyword(s):

Amino Acids ◽

Genetic Algorithms ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Genetically Modified Organisms ◽

Fitness Function ◽

Unnatural Amino Acid ◽

Standard Genetic Code ◽

Genetic Codes

In the last decades, researchers have proposed the use of genetically modified organisms that utilize unnatural amino acids, i.e., amino acids other than the 20 amino acids encoded in the standard genetic code. Unnatural amino acids have been incorporated into genetically engineered organisms for the development of new drugs, fuels and chemicals. When new amino acids are incorporated, it is necessary to modify the standard genetic code. Expanded genetic codes have been created without considering the robustness of the code. The objective of this work is the use of genetic algorithms (GAs) for the optimization of expanded genetic codes. The GA indicates which codons of the standard genetic code should be used to encode a new unnatural amino acid. The fitness function has two terms; one for robustness of the new code and another that takes into account the frequency of use of amino acids. Experiments show that, by controlling the weighting between the two terms, it is possible to obtain more or less amino acid substitutions at the same time that the robustness is minimized.

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