An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli

•A method for incorporation of unnatural amino acids in response to the AGGA codon is developed.•The method is based on an engineered orthogonal pair of aminoacyl-tRNA synthetase and tRNAUCCU.•The engineered tRNAUCCU improves the efficiency as well as the specificity of the method.•Knocking out a competitive tRNA gene of ArgW increases the AGGA suppression efficiency.•The method is demonstrated for several unnatural amino acids and for multiple AGGA sites.