Protein engineering is a comprehensive toolbox for the chemical modification of enzymes in particular, and for the expansion of molecular functional diversity in general. In recent decades, two different categories have become established for the engineering of proteins. These include the approach of directed evolution approaches on the one hand and the strategies of rational protein design on the other hand. In particular, the use of noncanonical amino acids to introduce new functionalities has gained importance in the engineers’ toolbox. These include isostructural analogues of canonical amino acids as well as molecules with reactivities that can provide sites for further protein modifications. In this study, we have presented a strategy for manipulating the protein biosynthesis machinery towards the incorporation of noncognate fluorinated substrates. In general, fluorinated amino acids are not genetically encoded. These mainly synthetic building block are valuable for the design of particularly stable protein folds and for targeting highly specific protein-protein interactions. Fluorine is small and has a very low polarizability and the strongest inductive effect among the chemical elements found on earth. Due to these unique stereoelectronic properties, fluorine substitution is advantageously used in protein and peptide design. In this context, the strategy of directed evolution was applied to construct isoleucyl-transfer ribonucleic acid synthetase libraries for the isoleucine AUA rare codon reassignment with small aliphatic fluorinated amino acids, such as L-trifluoroethylglycine, by random mutagenesis. A suitable screening plasmid containing a mutant of superfolder green fluorescent protein (sfGFP) as reporter protein and a modified isoleucine transfer ribonucleic acid (tRNA_UAU) from Escherichia coli was produced to create an enhanced molecular adaptor level for gene expression. However, the required selection strain could not be constructed by genome editing due to the complexity of essential gene modification. In the second part of the study, different reporter proteins were used in advanced design with noncanonical amino acids for improvement of their biophysical, chemical, and biological properties. A robust alkene-tagged sfGFP variant was obtained, which is a valuable target in medicinal chemistry. In addition, the residue-specific incorporation of proline analogues into green fluorescent protein (GFP) derivates ─ enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange ─ enables the study of the role of prolines in the typical β-barrel structure organization.
