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 co-expression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannascii TyrRS (MjTyrRS-Dopa), we altered the anti-codon of tyrosyl-tRNA amber suppressor into AUA (MjtRNA(Tyr) AUA ) to recgonize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNA(Tyr) 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 co-expression of Dopa-recognizing TyrRSs, dfp-3 have a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS co-expression. This article is protected by copyright. All rights reserved.