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Title: Adaptive evolution of genomically recoded Escherichia coli

Abstract

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 ( Escherichia coli 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 engineeringmore » 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.« less

Authors:
; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Harvard Univ., Cambridge, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); Amazon Web Services Cloud Credits for Research Program
OSTI Identifier:
1420357
Alternate Identifier(s):
OSTI ID: 1527125
Grant/Contract Number:  
FG02-02ER63445
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 115 Journal Issue: 12; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; adaptive evolution; recoded genome; synthetic biology; genetic code expansion; nonstandard amino acids

Citation Formats

Wannier, Timothy M., Kunjapur, Aditya M., Rice, Daniel P., McDonald, Michael J., Desai, Michael M., and Church, George M. Adaptive evolution of genomically recoded Escherichia coli. United States: N. p., 2018. Web. doi:10.1073/pnas.1715530115.
Wannier, Timothy M., Kunjapur, Aditya M., Rice, Daniel P., McDonald, Michael J., Desai, Michael M., & Church, George M. Adaptive evolution of genomically recoded Escherichia coli. United States. doi:10.1073/pnas.1715530115.
Wannier, Timothy M., Kunjapur, Aditya M., Rice, Daniel P., McDonald, Michael J., Desai, Michael M., and Church, George M. Tue . "Adaptive evolution of genomically recoded Escherichia coli". United States. doi:10.1073/pnas.1715530115.
@article{osti_1420357,
title = {Adaptive evolution of genomically recoded Escherichia coli},
author = {Wannier, Timothy M. and Kunjapur, Aditya M. and Rice, Daniel P. and McDonald, Michael J. and Desai, Michael M. and Church, George M.},
abstractNote = {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 ( Escherichia coli 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.},
doi = {10.1073/pnas.1715530115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 12,
volume = 115,
place = {United States},
year = {2018},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1073/pnas.1715530115

Citation Metrics:
Cited by: 6 works
Citation information provided by
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Figures / Tables:

Fig. 1 Fig. 1: Representative trajectories showing changes in fitness during the evolution of the four E. coli strains discussed. (A, Top Left; Parent) Two lineages of nonrecoded ECNR2 (engineered from E. coli MG1655 K-12). (A, Top Right; Recoded) Two lineages of recoded C321.ΔA (321 UAG→UAA and RF1). (A, Bottom Left; Recoded.ΔRF1)more » Four lineages of recoded C321.ΔA-v2 (C321.ΔA with engineered reversion of some off-target mutations that occurred during recoding). (A, Bottom Right; Recoded.ΔRF1-v2) Two lineages of recoded C321 (C321.ΔA with prfA gene restored). (B) Ancestral and final doubling time measurements sampled from all lineages of Parent (blue), Recoded (red), Recoded.ΔRF1 (yellow), and Recoded.ΔRF1v2 (green).« less

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