Optimized Directed Evolution of E. coli leucyl-tRNA Synthetase adds many Noncanonical Amino Acids into the Eukaryotic Genetic Code Including Ornithine and Nϵ-Acetyl-Methyllysine

IF 16.1 1区 化学 Q1 CHEMISTRY, MULTIDISCIPLINARY
Dr. Elise D. Ficaretta, Tarah J. Yared, Dr. Subrata Bhattacharjee, Lena A. Voss, Dr. Rachel L. Huang, Prof. Abhishek Chatterjee
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引用次数: 0

Abstract

Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in eukaryotes has predominantly relied on the pyrrolysyl-tRNA synthetase/tRNA pair. However, access to additional easily engineered pairs is crucial for expanding the structural diversity of the ncAA toolbox in eukaryotes. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair presents a particularly promising alternative. This pair has been engineered to charge a small yet structurally diverse group of ncAAs in eukaryotic cells. However, expanding the substrate scope of EcLeuRS has been difficult due to the suboptimal yeast-based directed evolution platform used for its engineering. In this study, we address this limitation by optimizing the yeast-based directed evolution platform for efficient selection of ncAA-selective EcLeuRS mutants. Using the optimized selection system, we demonstrate rapid isolation of many novel EcLeuRS mutants capable of incorporating various ncAAs in mammalian cells, including ornithine and Nϵ-acetyl-methyllysine, a recently discovered post-translational modification in mammalian cells.

Abstract Image

优化的大肠杆菌亮氨酸- trna合成酶定向进化在真核生物遗传密码中增加了许多非规范氨基酸,包括鸟氨酸和nε -乙酰基甲基赖氨酸
在真核生物中,将非典型氨基酸(ncAAs)特异性地结合到蛋白质中主要依赖于吡咯糖基-tRNA 合成酶/tRNA 对。然而,获得更多易于工程化的配对对于扩大真核生物 ncAA 工具箱的结构多样性至关重要。大肠杆菌衍生的亮氨酰-tRNA 合成酶(EcLeuRS)/tRNA 对是一个特别有前途的选择。这对合成酶已被设计用于真核细胞中一小部分结构多样的 ncAAs。然而,扩大 EcLeuRS 的底物范围一直很困难,原因是其工程化所用的酵母定向进化平台不够理想。在本研究中,我们通过优化基于酵母的定向进化平台来有效选择 ncAA 选择性 EcLeuRS 突变体,从而解决了这一局限性。利用优化后的选择系统,我们证明了可以快速分离出许多新型 EcLeuRS 突变体,这些突变体能够在哺乳动物细胞中整合各种 ncAAs,包括鸟氨酸和 Nε-乙酰甲基赖氨酸,这是最近在哺乳动物细胞中发现的一种翻译后修饰。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
26.60
自引率
6.60%
发文量
3549
审稿时长
1.5 months
期刊介绍: Angewandte Chemie, a journal of the German Chemical Society (GDCh), maintains a leading position among scholarly journals in general chemistry with an impressive Impact Factor of 16.6 (2022 Journal Citation Reports, Clarivate, 2023). Published weekly in a reader-friendly format, it features new articles almost every day. Established in 1887, Angewandte Chemie is a prominent chemistry journal, offering a dynamic blend of Review-type articles, Highlights, Communications, and Research Articles on a weekly basis, making it unique in the field.
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