Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins

Current experiments that rely on biosynthetic metabolic protein labeling with ¹⁹F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of ¹⁹F-labeled proteins as analyzed by ¹⁹F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.