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Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(TYr) Suppressor

机译：通过工程化的tRNA（TYr）抑制剂改善了非规范氨基酸的掺入

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The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA(TYr) cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA(Tyr) used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA(TYr) suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA(TYr) amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA(Tyr) used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid K-m values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA(Tyr) may enhance future incorporation of many ncAAs by engineered TyrRS enzymes.
机译：甲烷甲烷球菌酪氨酰-tRNA合成酶（TyrRS）：tRNA（TYr）同源对已用于将大量非规范氨基酸（ncAAs）掺入大肠杆菌的重组蛋白中。但是，尚未对这些实验中使用的抑制性tRNA（Tyr）的结构元件进行最佳性能研究。在这里，我们评估野生型詹氏甲烷球菌TyrRS和进化的3-硝基酪氨酰-tRNA合成酶（nitroTyrRS）对几种工程tRNA（TYr）抑制剂的稳态动力学参数，并将氨基酰化性质与寡糖的效率和保真度相关联。超级文件夹绿色荧光蛋白（sfGFP）的体内合成。最佳ncAA-sfGFP合成与tRNA（TYr）琥珀色抑制剂在反密码子环（G34C / G37A）中有两个取代，而D和可变环中的四个其他突变存在的tRNA（Tyr）中改善的氨酰化动力学相关在迄今为止的所有定向进化实验中，在体内和体外均有害。这些发现扩展到其他四个结合了不同ncAA的进化TyrRS酶中的三个。抑制性tRNA引起TyrRS和nitroTyrRS的氨基酸K-m值降低，这表明TyrRS的直接反密码子识别并不一定会妨碍该正交系统的优越性能，并为定向进化的新方法提供了见识。 G34C / G37A tRNA（Tyr）可能通过工程化的TyrRS酶增强未来许多ncAA的掺入。

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