Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, 310058, China.
Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.
Nat Commun. 2021 Dec 2;12(1):7039. doi: 10.1038/s41467-021-27399-x.
Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.
定点整合非天然氨基酸（UAAs）具有类似于天然氨基酸（NAAs）的整合效率和低背景活性，对于高效合成具有各种新化学功能的蛋白质和设计各种合成营养缺陷型非常有价值。然而，在文献中，这样高效的翻译系统仍然在很大程度上未知。在这里，我们描述了经过工程改造的嵌合苯丙氨酸系统，通过对氨酰-tRNA 合成酶及其相应的 tRNA 进行系统工程改造，极大地提高了携带 UAA 的蛋白质的产量。这些经过工程改造的合成酶/tRNA 对允许 UAA 的单点和多点掺入，效率与 NAA 相似，且具有高保真度。此外，使用进化的嵌合苯丙氨酸系统，我们构建了一系列严格依赖外源性供应 UAA 的大肠杆菌菌株。我们进一步表明，当补充 UAA 时，合成营养缺陷型细胞可以在活体小鼠中稳健生长。
