Enhanced production of Dopa-incorporated mussel adhesive protein using engineered translational machineries.

Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyr AUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyr AUA increased the production yield of Dopa-incorporated MAP foot protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.