Replacement of a histidine by a non-canonical amino acid to generate a dependent organism

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In a recent paper in JACS [1], Schultz and co-workers present a strategy where replacing a key amino acid of an enzyme by a non-standard amino acid (nsAA) produces an organism that requires this nsAA for survival. The amino acid they chose for this purpose was histidine (His), for two important reasons: (1) His residues are abundant in metalloproteins; and (2) the native His codons (CAU, CAC) differ at two positions from the amber codon, implying that two mutations would be required to revert the residue to His during an amber suppression experiment. They selected to introduce a nsAA residue in place of His in the metalloenzyme mannose-6-phosphate isomerase (ManA), which catalyzes the interconversion of D-mannose-6-phosphate (M6P) and D-fructose-6-phosphate (F6P). Using the aminoacyl-tRNA synthase HRS, which accepts both 3-methyl-L-histidine (MeH) and 3-(3-pyridyl)-alanine (PyA) but not His as a substrate, they could introduce MeH at position H264 of ManA. Subsequent analysis showed that that E. coli dependent on this ManA mutant displayed a growth defect. They subsequently improved its catalytic activity by directed evolution, finding that a single mutation, N262S, was sufficient to recover wild type activity. Interestingly, the reversion rate found was less than 10-11 escapees per colony forming unit, much lower than the acceptable value 10-8) for biocontainment required by the National Institutes of Health.

  1. Fei Gan, Renhe Liu, Feng Wang, and Peter G. Schultz (2018). Functional Replacement of Histidine in Proteins To Generate Noncanonical Amino Acid Dependent Organisms. JACS. doi:10.1021/jacs.7b13452

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