Jeong Ye Seul, Yang Byeongseon, Yang Byungseop, Shin Mincheol, Seong Jihyoun, Cha Hyung Joon, Kwon Inchan
School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea.
Department of Chemical Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea.
Biotechnol Bioeng. 2020 Jul;117(7):1961-1969. doi: 10.1002/bit.27339. Epub 2020 Apr 13.
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 (MjtRNA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNA 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.
贻贝粘附蛋白（MAPs）作为生物胶水具有巨大潜力，特别是在潮湿条件下。尽管在酪氨酸营养缺陷型大肠杆菌细胞中体内3,4-二羟基苯丙氨酸（多巴）的残基特异性掺入能够生产掺入多巴的生物工程化MAPs（dMAPs），但低产量阻碍了dMAPs的实际应用。dMAPs的这种低产量是由于非标准氨基酸多巴在大肠杆菌细胞中的翻译活性较低。在此，为了提高dMAPs的产量，我们研究了识别多巴的酪氨酰-tRNA合成酶（TyrRSs）的共表达。为了使用对多巴具有特异性的詹氏甲烷球菌TyrRS（MjTyrRS-Dopa），我们将酪氨酰-tRNA琥珀抑制子的反密码子改变为AUA（MjtRNA）以识别酪氨酸密码子（AUA）。MjTyrRS-Dopa和MjtRNA的共过表达使掺入多巴的MAP足部蛋白3型（dfp-3）的产量提高了57%。同样，大肠杆菌TyrRS（EcTyrRS）的过表达使dfp-3的产量提高了72%。即使共表达识别多巴的TyrRSs，与未共表达TyrRSs制备的dfp-3相比，dfp-3仍具有较高的多巴掺入率（超过90%）。
