Protein translation from the ribosome seemingly requires dozens of sophisticated aminoacyl-tRNA synthetases (aaRSs). Despite the discovery of tRNA-aminoacylating ribozymes, such as the flexizyme, synthesizing protein enzymes from highly simplified translation systems in the absence of aaRS remains undemonstrated. Here, we show the translation of multiple protein enzymes of distinct functions using solely flexizyme-charged tRNAs, through improving the translation yield by concentrating cation-depleted tRNAs. Notably, we used the aaRS-free translation system to produce an active aaRS (TrpRS), which in turn catalyzed the charging of more tRNAs. Moreover, toward realizing mirror-image translation, we performed the mirror-image tRNA charging with D-amino acids by a synthetic L-flexizyme. Our work demonstrates the feasibility of translating protein enzymes from a highly simplified translation apparatus without aaRS and drastically reduces the requirement to chemically synthesize dozens of large aaRS proteins for realizing mirror-image translation.

核糖体的蛋白质翻译似乎需要数十种复杂的氨酰基tRNA合成酶（aaRSs）。尽管发现了tRNA-氨基酰化核酶，如弹性酶，但在没有aaRS的情况下，从高度简化的翻译系统合成蛋白质酶的现象仍未被证实。在这里，我们展示了通过仅浓缩弹性酶的tRNA，通过浓缩阳离子贫乏的tRNA，提高翻译产量，即可翻译出具有不同功能的多种蛋白质酶。值得注意的是，我们使用了无aaRS的翻译系统来产生活性aaRS（TrpRS），进而催化更多tRNA的充电。此外，为了实现镜像翻译，我们通过合成的L-弹性酶对D-氨基酸进行了镜像tRNA充电。