Orthogonal translation enables heterologous ribosome engineering in E. coli

Kolber, Natalie S.; Fattal, Ranan; Bratulić, Siniša; Carver, Gavriela D.; Badran, Ahmed H.

doi:10.1038/s41467-020-20759-z

Cited by 26 publications

(19 citation statements)

References 65 publications

(52 reference statements)

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“…Cumulatively, our findings highlight significant qtRNA evolvability for efficient and amino acid-specific quadruplet codon translation, demonstrating essential properties necessary for the development of an exclusively quadruplet codon code. Through the extension of this directed evolutionary framework and the newly developed orthogonal multi-qtRNA expression plasmids, rRNA engineering 80 and evolution strategies may be necessary to eliminate crosstalk with triplet codons and maintain quadruplet coding frames. In particular, targeted engineering of ribosomal conformational may further improve decoding fidelity beyond the recently described +1 frameshifting during tRNA-mRNA translocation 22 .…”

Section: Discussionmentioning

confidence: 99%

Multiplex suppression of four quadruplet codons via tRNA directed evolution

et al. 2021

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Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon–anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

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Section: Discussionmentioning

confidence: 99%

Multiplex suppression of four quadruplet codons via tRNA directed evolution

et al. 2021

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“…However, the o-ribosome is defective and produces proteins with a tenfold lower capacity than that of the natural ribosome. To solve this problem, various optimization strategies have been applied to improve the orthogonal translation system in recent years [84,85]. Among them, Liu et al [84] utilized phage-assisted continuous evolution technology for rapid optimization of 16S rRNA by screening pressure.…”

Section: Decoupling Cell Growth and Recombinant Protein Productionmentioning

confidence: 99%

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

et al. 2022

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Escherichia coli, one of the most efficient expression hosts for recombinant proteins (RPs), is widely used in chemical, medical, food and other industries. However, conventional expression strains are unable to effectively express proteins with complex structures or toxicity. The key to solving this problem is to alleviate the host burden associated with protein overproduction and to enhance the ability to accurately fold and modify RPs at high expression levels. Here, we summarize the recently developed optimization strategies for the high-level production of RPs from the two aspects of host burden and protein activity. The aim is to maximize the ability of researchers to quickly select an appropriate optimization strategy for improving the production of RPs.

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“…While tethered ribosomes (17)(18)(19)(20) or cell-free strategies (21)(22)(23)(24)(25) can be used to identify functionally detrimental ribosomal mutations, efforts to build modified ribosomes remain hampered by practical considerations in making and evaluating rRNA libraries. For example, the combinatorial space for rRNA evolution is large, such that random mutagenesis and selection approaches cannot be feasibly used to screen all possible variants.…”

Section: Introductionmentioning

confidence: 99%

Computationally-guided design and selection of ribosomal active site mutants with high activity

Kofman

Watkins²,

Kim

et al. 2022

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Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g., library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design may serve as a powerful tool for high-resolution rRNA design.

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Orthogonal translation enables heterologous ribosome engineering in E. coli

Cited by 26 publications

References 65 publications

Multiplex suppression of four quadruplet codons via tRNA directed evolution

Multiplex suppression of four quadruplet codons via tRNA directed evolution

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

Computationally-guided design and selection of ribosomal active site mutants with high activity

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