Additional approaches to further improve the target production and OTS performance can be followed in both hosts: Besides improving the aminoacyl-tRNA synthetase efficiency, rational (re)design of orthogonal tRNAs can allow for more efficient ncAA incorporation (Fan et al., 2015; Maranhao and Ellington, 2017). Genome engineering can allow the knockout of RF-1, which otherwise competes with amber suppressors and leads to translation termination. Accordingly, the resulting E. coli K and B strains can enable efficient and multi-site ncAA incorporation (Mukai et al., 2015; Zheng et al., 2016). Moreover, elongation factor Tu (EF-Tu; which delivers the aminoacyl-tRNAs to the ribosome) and other parts the ribosomal machinery can be engineered (Mukai et al., 2017). Little is known about stop codon suppressor mutants in L. lactis. Only one instance of an amber suppressor was reported so far (Dickely et al., 1995). This unwanted mutation can in our experiments be excluded by the obtained mass spectrometry data. Additionally, with the first documented OTS shuttle vector system for E. coli, Salmonella enterica, and Vibrio cholerae, new developments also for Gram-positive species are expected in the long run (Volkwein et al., 2017).