To specifically introduce the ncAA at the desired position in the protein of interest, we focused on implementing the SCS method (Wang et al., 2001) in L. lactis. This technology allows reprogramming the amber stop codon TAG to a sense codon for the ncAA. Genome analysis revealed TAG to be the least frequently used stop codon in L. lactis (Gupta et al., 2004). Consequently, introduction of an OTS which is based on amber suppression was anticipated to have a low impact on host cell fitness. As key components, the pyrrolysine (Pyl) tRNA synthetase and its corresponding tRNA from the archaeon Methanosarcina mazei [pyrrolysyl-tRNA synthetase (PylRS)–tRNAPyl] are commonly utilized. This aminoacyl-tRNA synthetase recognizes its cognate tRNA and naturally charges it with the 22nd amino acid Pyl, allowing its ribosomal incorporation into the growing polypeptide chain. This enzyme is of particular interest in biotechnology because of its high substrate promiscuity resulting from a relatively unspecific amino acid recognition by mainly hydrophobic interactions, which allows incorporation of various new amino acid variants (Wan et al., 2014; Tharp et al., 2017). Being orthogonal in bacterial and eukaryotic cells, PylRS is commonly used for incorporation of ncAAs, not only in Escherichia coli, but also in yeast (Hancock et al., 2010) and mammalian cells (Mukai et al., 2008). Recently, SCS was established in Bacillus cereus as the first Gram-positive organism (Luo et al., 2016) and in Synechococcus elongatus as the first photoautotrophic cyanobacterium (Chemla et al., 2017).