Due to the complexity of the methodology, also the challenges of this method were analyzed in depth in E. coli. Previous studies gave insights into possible reasons for low production yields using an OTS: The kinetics of amino acid activation and tRNA charging by PylRS gave information about the efficacy of the enzyme. Relative to canonical aminoacyl tRNA synthetases, its kcat values are about 1000 times lower. In the natural context, this turnover rate of the enzyme is sufficient, since in Methanosarcina only around 50 Pyl (amber) codons are found in all transcribed genes. In contrast, in E. coli the leucyl-tRNA synthetase needs to provide substrates for approximately 150,000 codons. Relative to the already low efficiencies for the WT substrate Pyl, variants engineered for ncAA incorporation commonly perform worse (Guo et al., 2014). Certainly, SCS brings along a reduction in RiPP production yields, since five recombinant genes of different origins have to be expressed: pylTS from archaeal alongside nisABC from Gram-positive origin, all in a Gram-negative host organism. Combined with the metabolic burden to maintain the corresponding plasmids, this illustrates the challenge of combining SCS with RiPP synthesis (Ow et al., 2006).