An Engineered Methionyl-tRNA Synthetase Enables Azidonorleucine Incorporation In Methionine Auxotrophic and Prototrophic Bacteria

Unnatural amino acids can be incorporated into recombinant proteins in vivo through the use of engineered aminoacyl-tRNA synthetases (aaRSs). In studies conducted to screen methionyl-tRNA synthetase (MetRS) mutants that allow for the incorporation of the unnatural amino acid azidonorleucine (ANL), a MetRS variant with a leucine to glycine (L13G) mutation in its methionine binding pocket was identified. A methionine auxotrophic E. coli strain, M15MA, was engineered by replacing the genomic MetRS allele, metG, with the mutant synthetase gene, metG*, which encodes for the MetRS L13G variant. The resulting strain, M15MA metG*, was able to replace methionine with ANL at a 90% extent of incorporation. We have also transferred the metG* allele to a methionine prototrophic E. coli strain, MC1061. The physiological effects of the metG* mutation on MC1061 were characterized revealing that metG* endows MC1061 with a methionine auxotrophic phenotype. High levels (>20 mg/L culture) of ANL substituted protein were produced in MC1061 metG* with an extent of ANL incorporation of 42%, while protein made in wild-type MC1061 with a plasmid copy of metG* and a genomic copy of native metG only had a 17% ANL incorporation. Thus the MC1061 metG* strain can effectively incorporate ANL despite competition for the MetRS by endogenously biosynthesized Met. We anticipate that the MC1061 metG* host will find use as a host for recombinant proteins and in proteomic studies.