Acib - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria.
Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
Biotechnol J. 2019 Mar;14(3):e1800125. doi: 10.1002/biot.201800125. Epub 2018 Jun 11.
The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.
在琥珀终止密码子处特异性掺入非天然氨基酸（ncAA）需要氨酰-tRNA 合成酶和对应的琥珀终止密码子抑制 tRNA（tRNA）。已对产甲烷球菌的酪氨酸-tRNA 合成酶和产甲烷八叠球菌的吡咯赖氨酰-tRNA 合成酶（PylRS）进行了广泛的工程改造，以接受各种非天然氨基酸。来自脱硫脱硫弧菌的 PylRS/tRNA 对大肠杆菌是有功能的，然而，尚未报道该 PylRS 的变体。在这项研究中，作者描述了一种来自脱硫脱硫弧菌的细菌 PylRS，作者使用半理性方法对其进行了反应性 ncAA 对叠氮基-l-苯丙氨酸（DhAzFRS）的工程改造。DhAzFRS 优先选择对叠氮基-l-苯丙氨酸作为底物，而不是经典的 l-苯丙氨酸。此外，作者证明了携带 Methanosarcina mazei PylRS 的前 190 个 N 端氨基酸的杂种 DhAzFRS 在大肠杆菌中的功能。这些结果表明，细菌和古细菌的 PylRS 可以“混合搭配”以调整它们的底物特异性。
