Hu Liming, Qin Xuewen, Huang Yujia, Cao Wenbing, Wang Chuchen, Wang Yong, Ling Xinyu, Chen Heqi, Wu Dan, Lin Yu, Liu Tao
State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China.
College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China.
ACS Synth Biol. 2020 Oct 16;9(10):2723-2736. doi: 10.1021/acssynbio.0c00257. Epub 2020 Sep 23.
Genetic code expansion (GCE) is a powerful technique for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells, which is achieved through evolved aminoacyl-tRNA synthetase mutants. Stability is important for promoting enzyme evolution, and we found that many of the evolved synthetase mutants have reduced thermostabilities. In this study, we characterized two novel pyrrolysyl-tRNA synthetases (PylRSs) derived from thermophilic archaea: () and (). Further study demonstrated that the wild-type PylRSs and several mutants were orthogonal and active in both and mammalian cells and could thus be used for GCE. Compared with the commonly used PylRS, the wild-type thermophilic PylRSs displayed reduced GCE efficiency; however, some of the mutants, as well as some chimeras, outperformed their mesophilic counterparts in mammalian cell culture at 37 °C. Their better performance could at least partially be attributed to the fact that these thermophilic synthetases exhibit a threshold of enhanced stability against destabilizing mutations to accommodate structurally diverse substrate analogues. These were indicated by the higher melting temperatures (by 3-6 °C) and the higher expression levels that were typically observed for the PylRS and PylRS mutants relative to the equivalents. Using histone H3 as an example, we demonstrated that one of the thermophilic synthetase mutants promoted the incorporation of multiple acetyl-lysine residues in mammalian cells. The enzymes developed in this study add to the PylRS toolbox and provide potentially better scaffolds for PylRS engineering and evolution, which will be necessary to meet the increasing demands for expanded substrate repertoire with better efficiency and specificity in mammalian systems.
遗传密码扩展（GCE）是一种在活细胞中将非标准氨基酸（ncAAs）位点特异性掺入蛋白质的强大技术，这是通过进化的氨酰 - tRNA合成酶突变体实现的。稳定性对于促进酶的进化很重要，并且我们发现许多进化的合成酶突变体的热稳定性降低。在本研究中，我们表征了两种源自嗜热古菌的新型吡咯赖氨酸 - tRNA合成酶（PylRSs）：（）和（）。进一步的研究表明，野生型PylRSs和几个突变体在大肠杆菌和哺乳动物细胞中都是正交且有活性的，因此可用于GCE。与常用的PylRS相比，野生型嗜热PylRSs的GCE效率较低；然而，一些突变体以及一些嵌合体在37°C的哺乳动物细胞培养中表现优于它们的嗜温对应物。它们更好的性能至少部分可归因于这些嗜热合成酶对不稳定突变表现出增强稳定性的阈值，以适应结构多样的底物类似物。相对于大肠杆菌的等效物，PylRS和PylRS突变体通常观察到的较高解链温度（高3 - 6°C）和较高表达水平表明了这一点。以组蛋白H3为例，我们证明了一种嗜热合成酶突变体促进了哺乳动物细胞中多个乙酰赖氨酸残基的掺入。本研究中开发的酶增加了PylRS工具库，并为PylRS工程和进化提供了潜在更好的支架，这对于在哺乳动物系统中以更高的效率和特异性满足对扩展底物库不断增加的需求将是必要的。
