Abstract
Some extant organisms reassign the amber stop codon to a sense codon through evolution, and suppression of the amber codon with engineered tRNAs has been exploited to expand the genetic code for incorporating non-canonical amino acids (ncAAs) in live systems. However, it is unclear how the host cells respond and adapt to such amber suppression. Herein we suppressed the amber codon in Escherichia coli with an orthogonal tRNA/synthetase pair and cultured the cells under such a pressure for about 500 generations. We discovered that E. coli quickly counteracted the suppression with transposon insertion to inactivate the orthogonal synthetase. Persistent amber suppression evading transposon inactivation led to global proteomic changes with a notable up-regulation of a previously uncharacterized protein (YdiI) for which we identified an unexpected function of expelling plasmids. These results should be valuable for understanding codon reassignment in genetic code evolution and for improving the efficiency of ncAA incorporation.