An Engineered Methionyl‐tRNA Synthetase Enables Azidonorleucine Incorporation in Methionine Prototrophic Bacteria

The ability to endow recombinant proteins with novel chemical functionality by incorporating unnatural amino acids (UAAs) into proteins beyond the 20 natural amino acids has expanded our ability to directly control and engineer these biomolecules. The technique of adding UAAs to proteins can be performed in a residue-specific manner by reassigning a codon for one of the 20 natural amino acids to an UAA in a host that is auxotrophic for the natural amino acid substrate. 9] The simplest example of residue-specific incorporation occurs when the UAA is structurally analogous to a natural amino acid so that the host cell’s native translational machinery can be utilized. A larger set of UAAs has been incorporated into proteins by engineering the aminoacyl-tRNA synthetases (aaRSs), 14–17] which function to ensure the fidelity of amino acid incorporation during protein synthesis. In studies performed to identify methionyl-tRNA synthetase (MetRS) variants that allow for the incorporation of azidonorleucine (ANL), a MetRS variant containing a single leucine to glycine (L13G) amino acid change in its Met binding pocket was discovered. The MetRS L13G variant activates ANL and retains a diminished ability to activate Met with a 270-fold lower specificity constant. Residue-specific UAA incorporation studies with engineered aaRSs typically use auxotrophic E. coli strains that retain the wild-type copy of the aaRS gene on the chromosome and add the engineered aaRS gene on a multicopy plasmid. 19, 20] Previously, we engineered a Met auxotrophic E. coli strain with the native MetRS gene (metG) replaced with a single genomic copy of the MetRS L13G gene (metG*). Despite the attenuation of its MetRS activity toward Met, this strain grows normally when provided with Met and is able to produce high levels of recombinant protein containing ANL. This initial study focused on a Met auxotrophic strain in which the intracellular pool of Met is depleted, allowing ANL to be the preferred substrate for the MetRS L13G. In this work we investigate whether a single genomic copy of metG* can permit high levels of ANL incorporation in a Met prototrophic strain. In a Met prototroph, exogenously added ANL in the culture medium must compete with an intracellular pool of Met. Here, we have inserted the metG* allele into the Met prototroph MC1061, characterized the physiology of the resulting strain, and tested its ability to incorporate ANL into recombinant proteins. Previously, the metG* gene was introduced into the genome of a Met auxotrophic strain (M15MA) based on the method of Datsenko and Wanner for chromosomal knockouts. Since metG is an essential gene in E. coli, we simultaneously knocked out the chromosomal copy of metG and integrated the metG* gene to generate the strain M15MA metG* (Figure 1). Subse-