Engineering posttranslational proofreading to discriminate nonstandard amino acids

Kunjapur, Aditya M.; Stork, Devon A.; Kuru, Erkin; Vargas-Rodriguez, Oscar; Landon, Matthieu; Söll, Dieter; Church, George M.

doi:10.1073/pnas.1715137115

Cited by 38 publications

(51 citation statements)

References 58 publications

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“…To evaluate the utility of our evolved recoded strains for nsAA incorporation, we isolated clonal populations from three evolved Recoded.ΔRF1-v2 strains and cotransformed them with plasmids harboring an orthogonal translation system and a reporter protein. If the orthogonal translation system is sufficiently specific for an nsAA, then nsAA incorporation is proportional to full-length reporter protein formation based on suppression of UAG codons in the gene encoding the reporter protein (50).…”

Section: Evolved Recoded Strains Show High Specificity For Nsaa Incormentioning

confidence: 99%

Adaptive evolution of genomically recoded Escherichia coli

Wannier

Kunjapur

Rice

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Efforts are underway to construct several recoded genomes anticipated to exhibit multivirus resistance, enhanced nonstandard amino acid (nsAA) incorporation, and capability for synthetic biocontainment. Although our laboratory pioneered the first genomically recoded organism ( strain C321.∆A), its fitness is far lower than that of its nonrecoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for nsAA-linked applications requiring defined media, such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceeded the growth rates of both the ancestral C321.∆A and nonrecoded strains. We used next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstructed individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occurred only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared with application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.

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Section: Evolved Recoded Strains Show High Specificity For Nsaa Incormentioning

confidence: 99%

Adaptive evolution of genomically recoded Escherichia coli

Wannier

Kunjapur

Rice

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…In Ubr1, the binding sites for the internal and N degrons are far apart, distributed along the 1,140 N-terminal residues of the 1,950-amino acid-long protein (44). By contrast, ClpS is only 106 residues long, suggesting that the canonical binding region recognizing the N degron (16,20,21,47,48) may overlap the region necessary for recognition of substrates with an N-terminal methionine and large hydrophobic amino acids at the fourth position (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

The expanded specificity and physiological role of a widespread N-degron recognin

Gao

Yeom

Groisman

2019

Proc. Natl. Acad. Sci. U.S.A.

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All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacteriumSalmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

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“…Finally, the identity of the N‐terminal residue strongly influences the in vivo half‐life of proteins . Current studies have shown that ncAAs incorporated at the N terminus of proteins have varying effects on the stability of proteins in vivo . However, protein stability may be difficult to predict when more diverse molecules are used for initiation in vivo.…”

Section: Initiation With Noncanonical Amino Acids In Vivomentioning

confidence: 99%