Adaptive evolution of genomically recoded Escherichia coli

Wannier, Timothy M.; Kunjapur, Aditya M.; Rice, Daniel P.; McDonald, Michael J.; Desai, Michael M.; Church, George M.

doi:10.1073/pnas.1715530115

Title: Adaptive evolution of genomically recoded Escherichia coli

Journal Article · 13 February 2018 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.1715530115 · OSTI ID:1420357

Wannier, Timothy M. ^[1]; Kunjapur, Aditya M. ^[1]; Rice, Daniel P. ^[2]; McDonald, Michael J. ^[2]; Desai, Michael M. ^[2];

^[1]

Harvard Medical School, Boston, MA (United States)
Harvard Univ., Cambridge, MA (United States)

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 colistrain C321.ΔA), 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 introduce adaptive evolution of C321.ΔA 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.ΔA 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 work 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.ΔA strain.

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Research Organization:: Harvard Univ., Cambridge, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); Amazon Web Services Cloud Credits for Research Program

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 1420357

Alternate ID(s):: OSTI ID: 1527125

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, Issue 12; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 49 works

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