The expansion of the genetic code using orthogonal aminoacyl-tRNA synthetase/tRNA pairs is an established tool in synthetic biology. However, known PylRS-based systems suffer from low catalytic efficiency, especially in the multisite incorporation of ncAAs. This limits both yield and industrial applicability.

The present invention addresses this limitation by developing a highly active, psychrophilic PylRS variant from M. burtonii with targeted mutations in the catalytic centre. This makes it possible to specifically equip recombinant proteins with additional chemical functions that are not accessible or only very inefficiently accessible using conventional biotechnological methods. During protein production in the cell, the greatly improved enzyme ensures that so-called non-canonical amino acids (ncAAs) are reliably and efficiently incorporated into proteins. These ncAAs carry special chemical groups, e.g. for coupling reactions, labelling or light-active functions.

The efficiency of the technology has already been validated in E. coli and in human HEK293T cells.