Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue

Abstract

Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.Cellular growth in the presence of unnatural amino acids often results in at least the partial incorporation of the analogues into proteins. For example, although 4-fluorotryptophan (4fW) is eventually toxic to cells, bacteria can grow for several generations in its presence and proteins that contain high levels of this analogue can be isolated (5, 29). Similarly, p-Cl-phenylalanine has been shown to replace phenylalanine in vivo, but it drastically decreases luciferase activity (14). Azaleucine can also be partially incorporated in place of arginine and has been shown to suppress an otherwise lethal mutation in thymidylate synthase (20). It has recently been shown that a tRNA synthetase with compromised editing function will allow the replacement of valine with cysteine, threonine, or ␣-aminobutyrate (9). Similarly, a tyrosyl-tRNA synthetase mutant has been isolated which discriminates against azatyrosine less readily than the wild type (13). All of these examples demonstrate that the partial substitution of one amino acid for another is possible.Current methods for unnatural amino acid incorporation in vivo involve either the site-specific incorporation of an unnatural amino acid into a few proteins (11) or the partial incorporation into many proteins. In contrast, we and Wong (37) have attempted to incorporate unnatural amino acids throughout an organismal proteome. Such attempts to develop unnatural organisms should enhance our understanding of the extent to which even the most basic biochemistry is evolvable or optimal and may suggest metabolic alternatives for engineered organisms. In order to explore these possibilities, we attempted to evolve Escherichia coli variants which could survive on 4fW, which is otherwise toxic (5, 29).Evolutiona...

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…56 Similar experiments were attempted by Bacher and Ellington some years later (vide infra) but so far without success. 57,58 In 1988, Bronskill and Wong demonstrated that (4-F)Trp could not only be used in 19 F NMR analysis but also facilitates the identification of the contribution of chromophores, like Tyr or Phe, to the spectroscopic features of a protein as (4-F)Trp itself is a non-fluorescent Trp analog. 59 In addition, fluorinated Trp analogs have in general been commonly used to perform spectroscopic and unfolding studies (vide infra and e.g.…”

Gelatinase-mediated migration and invasion of cancer cells

“…Third, this method replaces a single amino acid rather than expands the amino acid repertoire. One additional issue of concern is that some amino acid analogues are toxic to the cells thus reducing their utility in protein expression, although tolerant mutant bacteria can be isolated (Bacher and Ellington, 2001). Of the many amino acid analogues that can be incorporated by misaminoacylation, fluorinated analogues of tyrosine, tryptophan, phenylanine, and leucine have been the most widely used (Hagen et al, 1978;Minks et al, 1999;Minks et al, 2000;Rennert and Anker, 1963).…”

Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase

“…In contrast, eight other tryptophan analogues had only a slight effect (24). When E. coli was grown under conditions of tryptophan analogue incorporation, almost no effect on growth rate occurred until the media carried a ratio of Ͼ97% analogue relative to the natural amino acid (25). A trifluorinated analogue of isoleucine caused growth rate inhibition in an isoleucine auxotroph (26).…”