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

► We produced an improved system for production of recombinant phosphoproteins in Escherichia coli. ► Genome modification and strain engineering of E. coli enabled RF1 deletion. ► Single and double phosphoserine residues were incorporated into recombinant proteins at enhanced efficiency. ► Phosphoserine insertion at native UAG sites reduces cellular fitness and viability. 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) [2]. However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.