Chemical Evolution of a Bacterial Proteome.

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

[1]  C. Yanofsky,et al.  Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli , 1991, Journal of bacteriology.

[2]  N. Budisa.  Xenobiology, New-to-Nature Synthetic Cells and Genetic Firewall , 2014 .

[3]  E. Ron,et al.  Changes in cell dimensions during amino acid starvation of Escherichia coli , 1982, Journal of bacteriology.

[4]  G. Cohen,et al.  Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur. , 1957, Biochimica et biophysica acta.

[5]  R. Huber,et al.  Proteins with β‐(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids , 2001, Protein science : a publication of the Protein Society.

[6]  Carlos G. Acevedo-Rocha,et al.  On the road towards chemically modified organisms endowed with a genetic firewall. , 2011, Angewandte Chemie.

[7]  U. Keyser,et al.  Indole Transport across Escherichia coli Membranes , 2011, Journal of bacteriology.

[8]  N. Budisa,et al.  Designing novel spectral classes of proteins with a tryptophan-expanded genetic code , 2004, Biological chemistry.

[9]  M. Yčas,et al.  Incorporation of the amino acid analog tryptazan into the protein of Escherichia coli. , 1957, Archives of biochemistry and biophysics.

[10]  Carlos G. Acevedo-Rocha,et al.  Auf dem Weg zu chemisch veränderten Organismen mit genetischer Firewall , 2011 .

[11]  P G Schultz,et al.  A general method for site-specific incorporation of unnatural amino acids into proteins. , 1989, Science.

[12]  S. Lok,et al.  Mutations Enabling Displacement of Tryptophan by 4-Fluorotryptophan as a Canonical Amino Acid of the Genetic Code , 2014, Genome biology and evolution.

[13]  D. Söll,et al.  UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota , 2013, Proceedings of the National Academy of Sciences.

[14]  R. Furter.  Expansion of the genetic code: Site‐directed p‐fluoro‐phenylalanine incorporation in Escherichia coli , 1998, Protein science : a publication of the Protein Society.

[15]  Gabriela R. Moura,et al.  Development of the genetic code: Insights from a fungal codon reassignment , 2010, FEBS letters.

[16]  Philippe Marlière,et al.  Chemical evolution of a bacterium's genome. , 2011, Angewandte Chemie.

[17]  D. Söll,et al.  Upgrading protein synthesis for synthetic biology. , 2013, Nature chemical biology.

[18]  Andrew D. Ellington,et al.  Selection and Characterization of Escherichia coliVariants Capable of Growth on an Otherwise Toxic Tryptophan Analogue , 2001, Journal of bacteriology.

[19]  J. Chin,et al.  Expanding and reprogramming the genetic code of cells and animals. , 2014, Annual review of biochemistry.

[20]  A. Pardee,et al.  Incorporation of azatryptophan into proteins of bacteria and bacteriophage. , 1956, Biochimica et biophysica acta.

[21]  T H Jukes,et al.  Evolutionary changes in the genetic code. , 1993, Comparative biochemistry and physiology. B, Comparative biochemistry.

[22]  C. Yanofsky,et al.  Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli , 1995, Journal of bacteriology.

[23]  C. W. Bird.  A new aromaticity index and its application to five-membered ring heterocycles , 1985 .

[24]  G. Zon,et al.  Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits. , 1987, The Journal of biological chemistry.

[25]  J. Wong,et al.  Membership mutation of the genetic code: loss of fitness by tryptophan. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Markus Schmidt,et al.  Xenobiology: A new form of life as the ultimate biosafety tool , 2010, BioEssays : news and reviews in molecular, cellular and developmental biology.

[27]  Philippe Marliere,et al.  The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world , 2009, Systems and Synthetic Biology.

[28]  Peter G. Schultz,et al.  Genomically Recoded Organisms Expand Biological Functions , 2013, Science.

[29]  C. Eckerskorn,et al.  High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. , 1995, European journal of biochemistry.