In comparative experiments using both bacterial hosts, currently, E. coli is still the preferable host, benefitting from years of SCS optimization. However, this can be changed in future given that the OTS efficiency in L. lactis can be optimized. As for the sophisticated E. coli systems which still hold room for optimization (Zheng et al., 2016), the genetic setup for the archaeal tRNA and its amino acyl tRNA synthetase expression certainly requires fine-tuning and balancing with the three nisin production genes. For example, in E. coli, pylTS were expressed constitutively and only nisABC expression was regulated by the inducible T7 promoter. This potentially led to a higher production yield of nisin(I4BocK) and nisin(K12BocK) in E. coli than in L. lactis. Additionally, the mass spectrometry data suggest a main population of fully post-translationally modified nisin variants in contrast to the mixed dehydration status resulting from L. lactis cultivation. In the latter host, the expression of pylTS as well as nisABTC was regulated by a PnisA promoter, a method optimized for RiPP synthesis in L. lactis. Therefore, different promoter setups and gene copy numbers for pylTS as well as RBS and codon optimization of pylS, which originates from a genetically distant archaeal host, are conceivable options. In previous works on B. cereus, the expression of the orthogonal tRNA was driven by host cell tRNA promoters – a promising setup to be tested using L. lactis (Luo et al., 2016).