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

被引：0
|
作者：
Wang, Fan [1 ,2 ]
Liu, Li-Hua [2 ,3 ,4 ]
Wang, Zhenyu [1 ]
Jiang, Ao [2 ]
Wu, Yi-Rui [2 ]
机构：
[1] Hainan Univ, Sch Trop Agr & Forestry, Haikou 570228, Hainan, Peoples R China
[2] Guangzhou Qianxiang Bioworks Co Ltd, Tidetron Bioworks Technol Guangzhou Co Ltd, Guangzhou 510000, Guangdong, Peoples R China
[3] Shantou Univ, Biol Dept, Coll Sci, Shantou 515063, Peoples R China
[4] Shantou Univ, Inst Marine Sci, Coll Sci, Shantou 515063, Peoples R China
来源：
NEW BIOTECHNOLOGY | 2024年 / 82卷
关键词：
Suppressor tRNAs; Premature termination codons; Directed evolution; Gradient biosensors; DIRECTED EVOLUTION; NONSENSE;
D O I：
10.1016/j.nbt.2024.05.005
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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 tRNATrp (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 tRNATrp (UGA) mutants exhibited over fivefold increases in readthrough efficiency.
引用
收藏
页码：85 / 91
页数：7
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