skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Refactoring the Genetic Code for Increased Evolvability

Abstract

ABSTRACT The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codesmore » allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.« less

Authors:
 [1];  [1]; ;  [1];
  1. Renewable and Sustainable Energy Institute, University of Colorado Boulder, Boulder, Colorado, USA, Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA
Publication Date:
Research Org.:
Univ. of Colorado, Boulder, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1437771
Alternate Identifier(s):
OSTI ID: 1506467
Grant/Contract Number:  
SC008812; SC0008812
Resource Type:
Published Article
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Name: mBio (Online) Journal Volume: 8 Journal Issue: 6; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Pines, Gur, Winkler, James D., Pines, Assaf, Gill, Ryan T., and Lee, ed., Sang Yup. Refactoring the Genetic Code for Increased Evolvability. United States: N. p., 2017. Web. doi:10.1128/mBio.01654-17.
Pines, Gur, Winkler, James D., Pines, Assaf, Gill, Ryan T., & Lee, ed., Sang Yup. Refactoring the Genetic Code for Increased Evolvability. United States. doi:10.1128/mBio.01654-17.
Pines, Gur, Winkler, James D., Pines, Assaf, Gill, Ryan T., and Lee, ed., Sang Yup. Tue . "Refactoring the Genetic Code for Increased Evolvability". United States. doi:10.1128/mBio.01654-17.
@article{osti_1437771,
title = {Refactoring the Genetic Code for Increased Evolvability},
author = {Pines, Gur and Winkler, James D. and Pines, Assaf and Gill, Ryan T. and Lee, ed., Sang Yup},
abstractNote = {ABSTRACT The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.},
doi = {10.1128/mBio.01654-17},
journal = {mBio (Online)},
number = 6,
volume = 8,
place = {United States},
year = {2017},
month = {11}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1128/mBio.01654-17

Citation Metrics:
Cited by: 2 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Addressing the Numbers Problem in Directed Evolution
journal, July 2008

  • Reetz, Manfred T.; Kahakeaw, Daniel; Lohmer, Renate
  • ChemBioChem, Vol. 9, Issue 11
  • DOI: 10.1002/cbic.200800298

Recent advances in synthetic biosafety
journal, January 2016


Codon Compression Algorithms for Saturation Mutagenesis
journal, September 2014

  • Pines, Gur; Pines, Assaf; Garst, Andrew D.
  • ACS Synthetic Biology, Vol. 4, Issue 5, p. 604-614
  • DOI: 10.1021/sb500282v

On the evolution of the genetic code.
journal, December 1965


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


Large-scale de novo DNA synthesis: technologies and applications
journal, April 2014

  • Kosuri, Sriram; Church, George M.
  • Nature Methods, Vol. 11, Issue 5
  • DOI: 10.1038/nmeth.2918

Expanding and Reprogramming the Genetic Code of Cells and Animals
journal, June 2014


Directed evolution of trimethoprim resistance in Escherichia coli
journal, April 2007


Genome engineering
journal, December 2009

  • Carr, Peter A.; Church, George M.
  • Nature Biotechnology, Vol. 27, Issue 12
  • DOI: 10.1038/nbt.1590

Evolving responsively: adaptive mutation
journal, July 2001

  • Rosenberg, Susan M.
  • Nature Reviews Genetics, Vol. 2, Issue 7
  • DOI: 10.1038/35080556

Early Fixation of an Optimal Genetic Code
journal, April 2000


Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides
journal, December 2003

  • Smith, H. O.; Hutchison, C. A.; Pfannkoch, C.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 26, p. 15440-15445
  • DOI: 10.1073/pnas.2237126100

Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm
journal, June 2009

  • Kumar, Prateek; Henikoff, Steven; Ng, Pauline C.
  • Nature Protocols, Vol. 4, Issue 7
  • DOI: 10.1038/nprot.2009.86

Altering the regioselectivity of cytochrome P450 BM-3 by saturation mutagenesis for the biosynthesis of indirubin
journal, November 2010


Metabolic engineering of microbial competitive advantage for industrial fermentation processes
journal, August 2016


Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli
journal, November 2015

  • Ho, Joanne M.; Reynolds, Noah M.; Rivera, Keith
  • ACS Synthetic Biology, Vol. 5, Issue 2, p. 163-171
  • DOI: 10.1021/acssynbio.5b00197

Exploring Nonnatural Evolutionary Pathways by Saturation Mutagenesis: Rapid Improvement of Protein Function
journal, December 1999

  • Miyazaki, Kentaro; Arnold, Frances H.
  • Journal of Molecular Evolution, Vol. 49, Issue 6
  • DOI: 10.1007/PL00006593

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

An Autotrophic Origin for the Coded Amino Acids is Concordant with the Coevolution Theory of the Genetic Code
journal, October 2016


The code within the codons
journal, January 1989


Quality control despite mistranslation caused by an ambiguous genetic code
journal, October 2008

  • Ruan, B.; Palioura, S.; Sabina, J.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 43
  • DOI: 10.1073/pnas.0809179105

Rewriting the Genetic Code
journal, September 2017


Robustness and Evolvability
journal, September 2010


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


Codon—anticodon pairing: The wobble hypothesis
journal, August 1966


Genome dynamics during experimental evolution
journal, October 2013

  • Barrick, Jeffrey E.; Lenski, Richard E.
  • Nature Reviews Genetics, Vol. 14, Issue 12
  • DOI: 10.1038/nrg3564

Plasmid-based one-pot saturation mutagenesis
journal, October 2016

  • Wrenbeck, Emily E.; Klesmith, Justin R.; Stapleton, James A.
  • Nature Methods, Vol. 13, Issue 11
  • DOI: 10.1038/nmeth.4029

Rewiring the keyboard: evolvability of the genetic code
journal, January 2001

  • Knight, Robin D.; Freeland, Stephen J.; Landweber, Laura F.
  • Nature Reviews Genetics, Vol. 2, Issue 1
  • DOI: 10.1038/35047500

Exploring protein fitness landscapes by directed evolution
journal, December 2009

  • Romero, Philip A.; Arnold, Frances H.
  • Nature Reviews Molecular Cell Biology, Vol. 10, Issue 12
  • DOI: 10.1038/nrm2805

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment
journal, October 2016

  • Biddle, Wil; Schmitt, Margaret A.; Fisk, John D.
  • Nucleic Acids Research
  • DOI: 10.1093/nar/gkw948

A quantitative measure of error minimization in the genetic code
journal, November 1991

  • Haig, David; Hurst, Laurence D.
  • Journal of Molecular Evolution, Vol. 33, Issue 5
  • DOI: 10.1007/BF02103132

On the Fundamental Nature and Evolution of the Genetic Code
journal, January 1966

  • Woese, C. R.; Dugre, D. H.; Dugre, S. A.
  • Cold Spring Harbor Symposia on Quantitative Biology, Vol. 31, Issue 0
  • DOI: 10.1101/SQB.1966.031.01.093

Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis: Saturation Mutagenesis of Horseradish Peroxidase
journal, September 2015

  • Asad, Sedigheh; Dastgheib, Seyed Mohammad Mehdi; Khajeh, Khosro
  • Biotechnology and Applied Biochemistry, Vol. 63, Issue 6
  • DOI: 10.1002/bab.1437

A general method for saturation mutagenesis of cloned DNA fragments
journal, July 1985


The Resistome: A Comprehensive Database of Escherichia coli Resistance Phenotypes
journal, July 2016

  • Winkler, James D.; Halweg-Edwards, Andrea L.; Erickson, Keesha E.
  • ACS Synthetic Biology, Vol. 5, Issue 12
  • DOI: 10.1021/acssynbio.6b00150

Chemical synthesis of the mouse mitochondrial genome
journal, October 2010

  • Gibson, Daniel G.; Smith, Hamilton O.; Hutchison, Clyde A.
  • Nature Methods, Vol. 7, Issue 11
  • DOI: 10.1038/nmeth.1515

Visualizing high error levels during gene expression in living bacterial cells
journal, June 2010

  • Meyerovich, M.; Mamou, G.; Ben-Yehuda, S.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 25
  • DOI: 10.1073/pnas.0912989107

Expanded Genetic Codes Create New Mutational Routes to Rifampicin Resistance in Escherichia coli
journal, May 2016

  • Hammerling, Michael J.; Gollihar, Jimmy; Mortensen, Catherine
  • Molecular Biology and Evolution, Vol. 33, Issue 8
  • DOI: 10.1093/molbev/msw094

Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis
journal, April 2000

  • Tang, Mengjia; Pham, Phuong; Shen, Xuan
  • Nature, Vol. 404, Issue 6781
  • DOI: 10.1038/35010020

Balancing Robustness and Evolvability
journal, December 2006


Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
journal, May 2010


Order in the genetic code.
journal, July 1965

  • Woese, C. R.
  • Proceedings of the National Academy of Sciences, Vol. 54, Issue 1
  • DOI: 10.1073/pnas.54.1.71

The Genetic Code Is One in a Million
journal, September 1998

  • Freeland, Stephen J.; Hurst, Laurence D.
  • Journal of Molecular Evolution, Vol. 47, Issue 3
  • DOI: 10.1007/PL00006381

The RNA Code and Protein Synthesis
journal, January 1966

  • Nirenberg, M.; Caskey, T.; Marshall, R.
  • Cold Spring Harbor Symposia on Quantitative Biology, Vol. 31, Issue 0
  • DOI: 10.1101/SQB.1966.031.01.008

The genetic code constrains yet facilitates Darwinian evolution
journal, June 2013

  • Firnberg, Elad; Ostermeier, Marc
  • Nucleic Acids Research, Vol. 41, Issue 15
  • DOI: 10.1093/nar/gkt536

Computational method to reduce the search space for directed protein evolution
journal, March 2001

  • Voigt, C. A.; Mayo, S. L.; Arnold, F. H.
  • Proceedings of the National Academy of Sciences, Vol. 98, Issue 7
  • DOI: 10.1073/pnas.051614498

Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes
journal, October 2015

  • Cahn, J. K. B.; Baumschlager, A.; Brinkmann-Chen, S.
  • Protein Engineering Design and Selection
  • DOI: 10.1093/protein/gzv057

The Genome Project-Write
journal, June 2016


Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing
journal, May 2012

  • Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan
  • Nature Biotechnology, Vol. 30, Issue 6
  • DOI: 10.1038/nbt.2214

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

Massively parallel single-amino-acid mutagenesis
journal, January 2015

  • Kitzman, Jacob O.; Starita, Lea M.; Lo, Russell S.
  • Nature Methods, Vol. 12, Issue 3
  • DOI: 10.1038/nmeth.3223

The Molecular Diversity of Adaptive Convergence
journal, January 2012

  • Tenaillon, O.; Rodriguez-Verdugo, A.; Gaut, R. L.
  • Science, Vol. 335, Issue 6067, p. 457-461
  • DOI: 10.1126/science.1212986

Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement
journal, July 2011

  • Isaacs, Farren J.; Carr, Peter A.; Wang, Harris H.
  • Science, Vol. 333, Issue 6040, p. 348-353
  • DOI: 10.1126/science.1205822

Breaking the Degeneracy of the Genetic Code
journal, June 2003

  • Kwon, Inchan; Kirshenbaum, Kent; Tirrell, David A.
  • Journal of the American Chemical Society, Vol. 125, Issue 25
  • DOI: 10.1021/ja0350076

Selection, history and chemistry: the three faces of the genetic code
journal, June 1999

  • Knight, Robin D.; Freeland, Stephen J.; Landweber, Laura F.
  • Trends in Biochemical Sciences, Vol. 24, Issue 6
  • DOI: 10.1016/S0968-0004(99)01392-4

The dinB Gene Encodes a Novel E. coli DNA Polymerase, DNA Pol IV, Involved in Mutagenesis
journal, August 1999


Protein mistranslation: friend or foe?
journal, August 2014

  • Ribas de Pouplana, Liuís; Santos, Manuel A. S.; Zhu, Jun-Hao
  • Trends in Biochemical Sciences, Vol. 39, Issue 8
  • DOI: 10.1016/j.tibs.2014.06.002

Better prediction of functional effects for sequence variants
journal, June 2015


A Co-Evolution Theory of the Genetic Code
journal, May 1975

  • Wong, J. T. -F.
  • Proceedings of the National Academy of Sciences, Vol. 72, Issue 5
  • DOI: 10.1073/pnas.72.5.1909

A Comprehensive, High-Resolution Map of a Gene’s Fitness Landscape
journal, February 2014

  • Firnberg, Elad; Labonte, Jason W.; Gray, Jeffrey J.
  • Molecular Biology and Evolution, Vol. 31, Issue 6
  • DOI: 10.1093/molbev/msu081

FACS-optimized mutants of the green fluorescent protein (GFP)
journal, January 1996


Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis
journal, November 2015

  • Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang
  • Analytical and Bioanalytical Chemistry, Vol. 408, Issue 3
  • DOI: 10.1007/s00216-015-9181-5

ViennaRNA Package 2.0
journal, November 2011

  • Lorenz, Ronny; Bernhart, Stephan H.; Höner zu Siederdissen, Christian
  • Algorithms for Molecular Biology, Vol. 6, Issue 1
  • DOI: 10.1186/1748-7188-6-26

The Genetic code and Error Transmission
journal, October 1969

  • Alff-Steinberger, C.
  • Proceedings of the National Academy of Sciences, Vol. 64, Issue 2
  • DOI: 10.1073/pnas.64.2.584

Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354
journal, October 1996

  • White, Peter J.; Squirrell, David J.; Arnaud, Phillipe
  • Biochemical Journal, Vol. 319, Issue 2
  • DOI: 10.1042/bj3190343

Evolvability as a Function of Purifying Selection in TEM-1 β-Lactamase
journal, February 2015


Adding New Chemistries to the Genetic Code
journal, June 2010


MODOMICS: a database of RNA modification pathways
journal, January 2006

  • Dunin-Horkawicz, S.
  • Nucleic Acids Research, Vol. 34, Issue 90001
  • DOI: 10.1093/nar/gkj084

The origin of the genetic code
journal, December 1968


Evolutionary paths to antibiotic resistance under dynamically sustained drug selection
journal, December 2011

  • Toprak, Erdal; Veres, Adrian; Michel, Jean-Baptiste
  • Nature Genetics, Vol. 44, Issue 1, p. 101-105
  • DOI: 10.1038/ng.1034

Design and synthesis of a minimal bacterial genome
journal, March 2016


Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes
journal, April 2007

  • Reetz, Manfred T.; Carballeira, José Daniel
  • Nature Protocols, Vol. 2, Issue 4
  • DOI: 10.1038/nprot.2007.72

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing
journal, September 2012

  • Lee, H.; Popodi, E.; Tang, H.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 41
  • DOI: 10.1073/pnas.1210309109

Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N -Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB
journal, April 2014

  • Winkler, James D.; Garcia, Carlos; Olson, Michelle
  • Applied and Environmental Microbiology, Vol. 80, Issue 12
  • DOI: 10.1128/AEM.00499-14

Simulated evolution applied to study the genetic code optimality using a model of codon reassignments
journal, February 2011


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

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli
journal, April 2016

  • Bassalo, Marcelo C.; Garst, Andrew D.; Halweg-Edwards, Andrea L.
  • ACS Synthetic Biology, Vol. 5, Issue 7
  • DOI: 10.1021/acssynbio.5b00187

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

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering
journal, December 2016

  • Garst, Andrew D.; Bassalo, Marcelo C.; Pines, Gur
  • Nature Biotechnology, Vol. 35, Issue 1, p. 48-55
  • DOI: 10.1038/nbt.3718

Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds
journal, July 2004

  • Rui, Lingyun; Reardon, Kenneth F.; Wood, Thomas K.
  • Applied Microbiology and Biotechnology, Vol. 66, Issue 4
  • DOI: 10.1007/s00253-004-1698-z

Defining synonymous codon compression schemes by genome recoding
journal, October 2016

  • Wang, Kaihang; Fredens, Julius; Brunner, Simon F.
  • Nature, Vol. 539, Issue 7627
  • DOI: 10.1038/nature20124

The Impact of Message Mutation on the Fitness of a Genetic Code
journal, May 2002


Innate immune and chemically triggered oxidative stress modifies translational fidelity
journal, November 2009

  • Netzer, Nir; Goodenbour, Jeffrey M.; David, Alexandre
  • Nature, Vol. 462, Issue 7272
  • DOI: 10.1038/nature08576