(71d) Robustness of an E. coli Strain with a Defective Aminoacyl-tRNA Synthetase

A recombinant methionyl-tRNA synthetase (MetRS) has been previously engineered to incorporate unnatural amino acids via site-directed mutagenesis and screening based on cell-surface display [1]. MetRS containing a single amino acid mutation in the methionine binding pocket, leucine 13 to glycine (L13G), is very efficient at incorporating the unnatural amino acid azidonorleucine (ANL) into recombinant proteins. In addition, this mutant MetRS still retains the ability to incorporate methionine, although at a 270-fold lower rate than wild-type MetRS. Using the method of Datsenko and Wanner [2], the gene encoding this mutant MetRS, which we term metG*, has been introduced into the genome of a methionine auxotrophic strain, M15MA, replacing its wild-type metG allele. The metG* allele has also been introduced into the non-auxotrophic strains MC1061 and MC4100 via generalized transduction. Due to the fact that this mutant MetRS can affect each and every protein in the proteome, the goal of this current research has been to characterize the effects of this mutant MetRS on E. coli physiology. Growth behavior of the auxotrophic and non-auxotrophic strains has been assessed under a variety of conditions, such as growth temperature and methionine starvation and indicate that strains carrying the metG* allele are surprisingly robust. Here we demonstrate that a single genomic copy of metG* is capable of supporting robust cell growth and enabling the high-level expression and production of recombinant proteins containing ANL. Since this mutation has been introduced into the genome, as opposed to a plasmid-borne copy, M15MA metG* is genetically stable and does not require antibiotic selection to maintain the allele. We show the effectiveness of the M15MA metG* strain in producing azide-labeled proteins for bioconjugation via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Cells expressing biotinylated outer membrane protein C (OmpC) containing ANL (OmpC-ANL) and stained with fluorescent streptavidin exhibited a five-fold increase in median fluorescence relative to cells expressing OmpC-Met by flow cytometry measurements. Moreover, high yields and extent of incorporation of another recombinant protein, dihydrofolate reductase (DHFR), was achieved. The extent of ANL incorporation in DHFR-ANL produced from M15MA metG* is 90 %. M15MA metG* may enable further unnatural amino acid studies of this type as well as more fundamental investigations into the robustness of cellular protein synthesis.

[1] Link, A.J. et al., Proc Natl Acad Sci U S A. 2006 July 5; 103(27): 10180?10185.

[2] Datsenko, K.A. and B.L. Wanner, Proc Natl Acad Sci U S A. 2000 June 6; 97(12): 6640-6645.