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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 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 recodedmore » 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.« less

Authors:
 [1];  [1];  [2];  [2];  [2]; ORCiD logo [1]
+ Show Author Affiliations
  1. Department of Genetics, Harvard Medical School, Boston, MA 02115,
  2. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138
Publication Date:
Research Org.:
Harvard Univ., Cambridge, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); 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.
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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. https://doi.org/10.1073/pnas.1715530115
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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. https://doi.org/10.1073/pnas.1715530115.
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@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 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.},
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 = {Tue Feb 13 00:00:00 EST 2018},
month = {Tue Feb 13 00:00:00 EST 2018}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1073/pnas.1715530115
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Cited by: 49 works
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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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Works referenced in this record:

The Concentration of Polypeptide Chain Release Factors 1 and 2 at Different Growth Rates of Escherichia coli
journal, May 1994

  • Adamski, Frances M.; McCaughan, Kim K.; Jørgensen, Flemming
  • Journal of Molecular Biology, Vol. 238, Issue 3
  • DOI: 10.1006/jmbi.1994.1293

Expression of peptide chain release factor 2 requires high-efficiency frameshift
journal, July 1986

  • Craigen, William J.; Caskey, C. Thomas
  • Nature, Vol. 322, Issue 6076
  • DOI: 10.1038/322273a0

Design, synthesis, and testing toward a 57-codon genome
journal, August 2016

Addition of the keto functional group to the genetic code of Escherichia coli
journal, December 2002

  • Wang, L.; Zhang, Z.; Brock, A.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 1, p. 56-61
  • DOI: 10.1073/pnas.0234824100

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids
journal, January 2011

  • Ugwumba, Isaac N.; Ozawa, Kiyoshi; Xu, Zhi-Qiang
  • Journal of the American Chemical Society, Vol. 133, Issue 2
  • DOI: 10.1021/ja106416g

A Genetically Encoded Bidentate, Metal-Binding Amino Acid
journal, December 2007

  • Xie, Jianming; Liu, Wenshe; Schultz, Peter G.
  • Angewandte Chemie International Edition, Vol. 46, Issue 48
  • DOI: 10.1002/anie.200703397

Laboratory divergence of Methylobacterium extorquens AM1 through unintended domestication and past selection for antibiotic resistance
journal, January 2014

  • Carroll, Sean; Xue, Katherine S.; Marx, Christopher J.
  • BMC Microbiology, Vol. 14, Issue 1
  • DOI: 10.1186/1471-2180-14-2

RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media
journal, November 2010

  • Conrad, T. M.; Frazier, M.; Joyce, A. R.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 47
  • DOI: 10.1073/pnas.0911253107

The signal for the termination of protein synthesis in procaryotes
journal, January 1990

  • Brown, Chris M.; Stockwell, Peter A.; Trotman, Clive N. A.
  • Nucleic Acids Research, Vol. 18, Issue 8
  • DOI: 10.1093/nar/18.8.2079

The frequency of errors in protein biosynthesis
journal, August 1972

  • Loftfield, R. B.; Vanderjagt, D.
  • Biochemical Journal, Vol. 128, Issue 5
  • DOI: 10.1042/bj1281353

Programming cells by multiplex genome engineering and accelerated evolution
journal, July 2009

  • Wang, Harris H.; Isaacs, Farren J.; Carr, Peter A.
  • Nature, Vol. 460, Issue 7257, p. 894-898
  • DOI: 10.1038/nature08187

A novel class of bacterial translation factor RF3 mutations suggests specific structural domains for premature peptidyl-tRNA drop-off
journal, December 2009

Protein tRNA Mimicry in Translation Termination
journal, January 2001

  • Nakamura, Y.; Uno, M.; Toyoda, T.
  • Cold Spring Harbor Symposia on Quantitative Biology, Vol. 66, Issue 0
  • DOI: 10.1101/sqb.2001.66.469

RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites
journal, September 2011

  • Johnson, David B. F.; Xu, Jianfeng; Shen, Zhouxin
  • Nature Chemical Biology, Vol. 7, Issue 11
  • DOI: 10.1038/nchembio.657

Use of Adaptive Laboratory Evolution To Discover Key Mutations Enabling Rapid Growth of Escherichia coli K-12 MG1655 on Glucose Minimal Medium
journal, October 2014

  • LaCroix, Ryan A.; Sandberg, Troy E.; O'Brien, Edward J.
  • Applied and Environmental Microbiology, Vol. 81, Issue 1
  • DOI: 10.1128/AEM.02246-14

A Genetically Encoded Fluorescent Amino Acid
journal, July 2006

  • Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.
  • Journal of the American Chemical Society, Vol. 128, Issue 27
  • DOI: 10.1021/ja062666k

Extending enzyme molecular recognition with an expanded amino acid alphabet
journal, February 2017

  • Windle, Claire L.; Simmons, Katie J.; Ault, James R.
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 10
  • DOI: 10.1073/pnas.1616816114

Coding-Sequence Determinants of Gene Expression in Escherichia coli
journal, April 2009

Improving Nature's Enzyme Active Site with Genetically Encoded Unnatural Amino Acids
journal, August 2006

  • Jackson, Jennifer C.; Duffy, Sean P.; Hess, Kenneth R.
  • Journal of the American Chemical Society, Vol. 128, Issue 34
  • DOI: 10.1021/ja061099y

Evolution of high mutation rates in experimental populations of E. coli
journal, June 1997

  • Sniegowski, Paul D.; Gerrish, Philip J.; Lenski, Richard E.
  • Nature, Vol. 387, Issue 6634
  • DOI: 10.1038/42701

Functional Genomics: Expression Analysis ofEscherichia coli Growing on Minimal and Rich Media
journal, October 1999

Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli
journal, August 2002

  • Chin, J. W.; Martin, A. B.; King, D. S.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 17, p. 11020-11024
  • DOI: 10.1073/pnas.172226299

A genetically encoded fluorescent amino acid
journal, June 2006

  • Summerer, D.; Chen, S.; Wu, N.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 26, p. 9785-9789
  • DOI: 10.1073/pnas.0603965103

Optimized clinical performance of growth hormone with an expanded genetic code
journal, May 2011

  • Cho, H.; Daniel, T.; Buechler, Y. J.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 22
  • DOI: 10.1073/pnas.1100387108

Rapid and Inexpensive Evaluation of Nonstandard Amino Acid Incorporation in Escherichia coli
journal, September 2016

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for tRNA nonsense suppressors
journal, January 1984

  • Ryd�n, S. M.; Isaksson, L. A.
  • MGG Molecular & General Genetics, Vol. 193, Issue 1
  • DOI: 10.1007/BF00327411

The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli
journal, October 2015

  • Reisch, Chris R.; Prather, Kristala L. J.
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep15096

Recombinant expression of selectively sulfated proteins in Escherichia coli
journal, October 2006

  • Liu, Chang C.; Schultz, Peter G.
  • Nature Biotechnology, Vol. 24, Issue 11
  • DOI: 10.1038/nbt1254

Codon reassignment in the Escherichia coli genetic code
journal, August 2010

  • Mukai, T.; Hayashi, A.; Iraha, F.
  • Nucleic Acids Research, Vol. 38, Issue 22
  • DOI: 10.1093/nar/gkq707

The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination
journal, September 2004

  • Xie, Jianming; Wang, Lei; Wu, Ning
  • Nature Biotechnology, Vol. 22, Issue 10
  • DOI: 10.1038/nbt1013

Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load
journal, December 2012

  • Wielgoss, S.; Barrick, J. E.; Tenaillon, O.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 1
  • DOI: 10.1073/pnas.1219574110

Release Factor One Is Nonessential in Escherichia coli
journal, May 2012

  • Johnson, David B. F.; Wang, Chong; Xu, Jianfeng
  • ACS Chemical Biology, Vol. 7, Issue 8
  • DOI: 10.1021/cb300229q

Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon
journal, May 2015

  • Mukai, Takahito; Hoshi, Hiroko; Ohtake, Kazumasa
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep09699

Biocontainment of genetically modified organisms by synthetic protein design
journal, January 2015

  • Mandell, Daniel J.; Lajoie, Marc J.; Mee, Michael T.
  • Nature, Vol. 518, Issue 7537
  • DOI: 10.1038/nature14121

Methylation of Bacterial Release Factors RF1 and RF2 Is Required for Normal Translation Termination in Vivo
journal, October 2007

  • Mora, Liliana; Heurgué-Hamard, Valérie; de Zamaroczy, Miklos
  • Journal of Biological Chemistry, Vol. 282, Issue 49
  • DOI: 10.1074/jbc.M706076200

Suppression of Temperature-sensitive Defects of Polypeptide Release Factors RF-1 and RF-2 by Mutations or by an Excess of RF-3 inEscherichia coli
journal, May 1996

  • Matsumura, Kiyoyuki; Ito, Koichi; Kawazu, Yoichi
  • Journal of Molecular Biology, Vol. 258, Issue 4
  • DOI: 10.1006/jmbi.1996.0271

Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2
journal, January 1996

Genome evolution and adaptation in a long-term experiment with Escherichia coli
journal, October 2009

  • Barrick, Jeffrey E.; Yu, Dong Su; Yoon, Sung Ho
  • Nature, Vol. 461, Issue 7268
  • DOI: 10.1038/nature08480

Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli.
journal, June 1994

  • Mikuni, O.; Ito, K.; Moffat, J.
  • Proceedings of the National Academy of Sciences, Vol. 91, Issue 13
  • DOI: 10.1073/pnas.91.13.5798

Engineering posttranslational proofreading to discriminate nonstandard amino acids
journal, January 2018

  • Kunjapur, Aditya M.; Stork, Devon A.; Kuru, Erkin
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 3
  • DOI: 10.1073/pnas.1715137115

Design of a synthetic yeast genome
journal, March 2017

  • Richardson, Sarah M.; Mitchell, Leslie A.; Stracquadanio, Giovanni
  • Science, Vol. 355, Issue 6329
  • DOI: 10.1126/science.aaf4557

Mutagenesis and Evolution of Sulfated Antibodies Using an Expanded Genetic Code
journal, September 2009

  • Liu, Chang C.; Choe, Hyeryun; Farzan, Michael
  • Biochemistry, Vol. 48, Issue 37
  • DOI: 10.1021/bi9011429

Polypeptide release at sense and noncognate stop codons by localized charge-exchange alterations in translational release factors
journal, February 2002

  • Uno, M.; Ito, K.; Nakamura, Y.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 4
  • DOI: 10.1073/pnas.032457599

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale
journal, November 2006

  • Herring, Christopher D.; Raghunathan, Anu; Honisch, Christiane
  • Nature Genetics, Vol. 38, Issue 12
  • DOI: 10.1038/ng1906

Global Transcriptional Programs Reveal a Carbon Source Foraging Strategy by Escherichia coli
journal, February 2005

  • Liu, Mingzhu; Durfee, Tim; Cabrera, Julio E.
  • Journal of Biological Chemistry, Vol. 280, Issue 16
  • DOI: 10.1074/jbc.M414050200

Optimizing complex phenotypes through model-guided multiplex genome engineering
journal, May 2017

Total Synthesis of a Functional Designer Eukaryotic Chromosome
journal, March 2014

  • Annaluru, Narayana; Muller, Héloïse; Mitchell, Leslie A.
  • Science, Vol. 344, Issue 6179
  • DOI: 10.1126/science.1249252

Recoded organisms engineered to depend on synthetic amino acids
journal, January 2015

  • Rovner, Alexis J.; Haimovich, Adrian D.; Katz, Spencer R.
  • Nature, Vol. 518, Issue 7537
  • DOI: 10.1038/nature14095

Genomically Recoded Organisms Expand Biological Functions
journal, October 2013

  • Lajoie, M. J.; Rovner, A. J.; Goodman, D. B.
  • Science, Vol. 342, Issue 6156, p. 357-360
  • DOI: 10.1126/science.1241459

Design by Directed Evolution
journal, March 1998

  • Arnold, Frances H.
  • Accounts of Chemical Research, Vol. 31, Issue 3
  • DOI: 10.1021/ar960017f

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications
journal, September 2015

  • Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Hyeon Yoo, Tae
  • Biotechnology Journal, Vol. 10, Issue 12
  • DOI: 10.1002/biot.201500153
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