Orthogonal (O) ribosome-mediated translation of O-mRNAs enables the incorporation of up to three distinct non-canonical amino acids (ncAAs) into proteins in Escherichia coli (E. coli). However, the general and efficient incorporation of multiple distinct ncAAs by O-ribosomes requires scalable strategies for both creating efficiently and specifically translated O-mRNAs, and the compact expression of multiple O-aminoacyl-tRNA synthetase (O-aaRS)/O-tRNA pairs. We automate the discovery of O-mRNAs that lead to up to 40 times more protein, and are up to 50-fold more orthogonal, than previous O-mRNAs; protein yields from our O-mRNAs match or exceed those from wild-type mRNAs. These advances enable a 33-fold increase in yield for incorporating three distinct ncAAs. We automate the creation of operons for O-tRNA genes, and develop operons for O-aaRS genes. Combining our advances creates a 68-codon, 24-amino-acid genetic code to efficiently incorporate four distinct ncAAs into a single protein in response to four distinct quadruplet codons.

正交 (O) 核糖体介导的 O-mRNA 翻译能够将多达三种不同的非规范氨基酸 (ncAAs) 掺入大肠杆菌( E. coli）。然而，O-核糖体对多种不同的 ncAAs 的一般和有效结合需要可扩展的策略来创建有效和特异性翻译的 O-mRNAs，以及多个 O-氨酰-tRNA 合成酶 (O-aaRS)/O-tRNA 的紧凑表达对。我们自动发现 O-mRNA，与以前的 O-mRNA 相比，这些 O-mRNA 产生的蛋白质最多可增加 40 倍，正交性最多可增加 50 倍；我们的 O-mRNA 的蛋白质产量与野生型 mRNA 的产量相当或超过。这些进步使合并三种不同的 ncAA 的产量增加了 33 倍。我们自动创建 O-tRNA 基因的操纵子，并开发 O-aaRS 基因的操纵子。结合我们的进步创造了一个 68 个密码子，