title: Expanded cellular amino acid pools containing phosphoserine, phosphothreonine, and phosphotyrosine.
abstract: Adding nonstandard amino acids to the genetic code of E. coli 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 E. coli. We show that deletion of the phosphoserine phosphatase serB 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 serB 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.
authors.affiliation: Department of †Cellular & Molecular Physiology, ‡Systems Biology Institute, and §Molecular Biophysics & Biochemistry, Yale University , New Haven, Connecticut 06520, United States.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Steinfeld
authors.forename: Hans R
authors.initials: HR
authors.lastname: Aerni
authors.forename: Svetlana
authors.initials: S
authors.lastname: Rogulina
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Liu
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D019000:Phosphotyrosine / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D010744:Phosphoric Monoester Hydrolases; C022394:phosphoserine phosphatase; D000604:Amino Acyl-tRNA Synthetases