Expanded Cellular Amino Acid Pools Containing Phosphoserine, Phosphothreonine, and Phosphotyrosine

Adding nonstandard amino acids to the genetic code of <i>E. coli</i> expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accomplished, conditions that modulate intracellular phosphoamino acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in <i>E. coli</i>. We show that deletion of the phosphoserine phosphatase <i>serB</i> elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were grown in LB with supplemented phosphothreonine and <i>serB</i> deficient cells were grown in low phosphate media with supplemented phosphotyrosine, respectively. These findings remove a major barrier for further expansion of the genetic code with additional phosphorylated amino acids

Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion

a b s t r a c t Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151