A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli

IF 4.5 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

New biotechnology Pub Date : 2024-05-20 DOI:10.1016/j.nbt.2024.05.005

Fan Wang , Li-Hua Liu , Zhenyu Wang , Ao Jiang , Yi-Rui Wu

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引用次数: 0

Abstract

Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNA^{Trp (UGG)} reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNA^{Trp (UGA)} mutants exhibited over fivefold increases in readthrough efficiency.

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梯度噬菌体辅助连续进化法筛选大肠杆菌中的抑制性 tRNA

抑制性 tRNA 具有读取终止密码子的能力，同时还能维持正常的肽合成，因此在治疗由过早终止密码子（PTC）引起的疾病方面大有可为。然而，由于缺乏有效的抑制性 tRNAs 工程方法，限制了其应用潜力。在这里，我们介绍了一种定向进化技术，它采用了噬菌体辅助连续进化（PACE）技术，并结合了以 M13 基因 III 中各种 PTC 为特征的梯度生物传感器。利用这种新方法，我们成功地在大肠杆菌中进化出了通过 UGA 终止密码子读取的 tRNATrp (UGG)。大规模平行测序显示，这些突变主要发生在反密码子环中。最后，两个抑制 tRNATrp (UGA) 突变体的读取通过效率提高了五倍以上。

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

New biotechnology 生物-生化研究方法

CiteScore

11.40

自引率

1.90%

发文量

审稿时长

1 months

期刊介绍： New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international. The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.