| The universal genetic code encoding twenty canonical amino acids has remained invariant over possibly the past two billion years other than in limited changes in codon assignments in some genomes. This has raised the question of whether the code is in fact frozen and immutable. The basic mutability of the code was first proven when mutants of Bacillus subtilis which could encode 4-fluorotryptophan (4FT) in replacement of Tryptophan (Trp), or even completely reject Trp itself as an incompetent analogue, in the ensemble of encoded amino acids capable of supporting indefinite cell growth, were isolated. In the present study, the LC33 mutant of B. subtilis Trp-auxotroph QB928 has been further mutated to enable the encoding of 5-fluoroTryptophan (5FT) and 6-fluoroTryptophan (6FT) for indefinite growth. Mutants unable to grow on Trp could readily be back mutated to regain this capacity. Withdrawal of individual amino acids, nucleosides and other growth factors showed that most withdrawals did not prevent LC33 growth on 4FT. These observations, along with similar observations on growth supported by 5FT or 6FT suggest that the barrier against the admission of novel amino acid analogues into the genetically encoded amino acid ensemble is controlled by only a small number of proteins where the function of the analogue is particularly inadequate. The mutation of the genetic code to admit an amino acid analogue even as toxic as 5FT would thus require overcoming the functional bias of only a small number of proteins. Therefore a continued opening of the genetic code to a wide range of amino acid analogues for indefinite growth and protein engineering appears feasible through the mutational removal of such oligogenic barriers to evolution and change. |
