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.