The bacteria-derived tyrosyl-tRNA synthetase (TyrRS)/tRNA pair was first used for unnatural amino acid (Uaa) mutagenesis in eukaryotic cells over 15 years ago. It provides an ideal platform to genetically encode numerous useful Uaas in eukaryotes. However, this pair has been engineered to charge only a small collection of Uaas to date. Development of Uaa-selective variants of this pair has been limited by technical challenges associated with a yeast-based directed evolution platform, which is currently required to alter its substrate specificity. Here we overcome this limitation by enabling its directed evolution in an engineered strain ofE. coli(ATMY), where the endogenous TyrRS/tRNA pair has been functionally replaced with an archaeal counterpart. The facileE. coli-based selection system enabled rapid engineering of this pair to develop variants that selectively incorporate various Uaas, including p-boronophenylalanine, into proteins expressed in mammalian cells as well as in the ATMY strain ofE. coli.

中文翻译：

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复活细菌酪氨酰-tRNA合成酶/ tRNA对，以扩展大肠杆菌和真核生物的遗传密码

细菌衍生的酪氨酰-tRNA合成酶（TyrRS）/ tRNA对在15年前首次用于真核细胞中的非天然氨基酸（Uaa）诱变。它提供了一个理想的平台，可以对真核生物中大量有用的Uaa进行基因编码。但是，到目前为止，这对产品仅能收取少量的Uaas费用。该对的Uaa-选择性变体的开发受到与基于酵母的定向进化平台相关的技术挑战的限制，目前基于酵母的定向进化平台需要改变其底物特异性。在这里，我们通过使其在工程改造的E菌株中定向进化来克服了这一局限性。大肠杆菌（ATMY），其中内源性TyrRS / tRNA对已在功能上被古细菌对应物取代。方便。基于大肠埃希菌的选择系统使这对细菌能够快速工程化，从而开发出将多种Uaa（包括对-硼烷苯丙氨酸）选择性掺入哺乳动物细胞以及ATMY E株中表达的蛋白质的变体。大肠杆菌。