Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approaches have been used to modify ribosomes, EF-Tu, and tRNAs. Here, we provide an overview of these in vitro methods that are aimed at optimal expansion of the npAA repertoire and their applications for the development of de novo bioactive peptides containing various npAAs.

中文翻译：

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用于扩展可用非规范氨基酸的体外遗传密码重编程

遗传密码重编程使我们能够在体外通过核糖体将各种非蛋白氨基酸 (npAA) 掺入肽中。可用的 npAA 库已得到扩展，不仅包括具有非规范侧链的 l-α-氨基酸，还包括具有非规范主链的氨基酸。尽管npAA的单次掺入取得了成功，但多次和连续掺入常常效率低下，甚至不成功。为了克服这一障碍，人们使用工程方法来修饰核糖体、EF-Tu 和 tRNA。在这里，我们概述了这些旨在优化扩展 npAA 库的体外方法及其在开发含有各种 npAA 的从头生物活性肽中的应用。