Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

尽管遗传密码扩展技术在调节蛋白质的结构和功能方面前景广阔，但在大多数情况下需要外部添加 ncAAs，这通常限制了遗传密码扩展技术的实用性，尤其是对于膜内化较差的非规范氨基酸 (ncAAs) . 在这里，我们报告了原核和真核自主细胞的创建，这些细胞具有生物合成和基因编码磺基酪氨酸 (sTyr) 的能力，磺基酪氨酸是一种具有低膜渗透性的重要蛋白质翻译后修饰。与文献中报道的外源性喂养最高水平 sTyr 的细胞相比，这些工程化细胞能够以更高的产量产生位点特异性硫酸化蛋白质。我们使用这些自主细胞来制备具有位点特异性硫酸化作用的高效凝血酶抑制剂。