By transplanting identity elements into E. coli tRNAfMet , we have engineered an orthogonal initiator tRNA (itRNATy2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

中文翻译：

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体内非标准氨基酸的蛋白质合成起始。

通过将同一性元素移植到大肠杆菌tRNAfMet中，我们设计了一个正交启动子tRNA（itRNATy2），它是詹氏甲烷球菌TyrRS的底物。我们证明itRNATy2可以启动体内带有多种侧链的芳香族非规范氨基酸（ncAAs）的翻译。尽管最初的系统产量低，但是从基因组中删除tRNAfMet的多余副本提供了一种大肠杆菌菌株，其中非规范起始效率等同于延伸率。通过这种改进的系统，我们生产了在第一和第二位置包含两个不同ncAA的蛋白质，这是朝着在体内生产完全非天然多肽的第一步。这项工作为合成生物学提供了宝贵的工具，并证明了E具有非凡的多功能性。