The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface-displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthrough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N- and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a “drop-in” version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems—and protein translation apparatuses—to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.

中文翻译：

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记者系统体系结构影响非典型氨基酸掺入效率和保真度的测量

对蛋白质内非规范氨基酸（ncAA）进行基因编码的能力支持从基础生物学研究到增强生物疗法特性的越来越多的应用。目前，我们对ncAA掺入系统的定量理解被用于量化ncAA掺入事件的多种表征和分析方法所混淆。尽管有几种有效的报告系统支持这种测量，但尚不清楚来自不同报告器的定量结果如何相互关联，或者哪些细节对测量的影响最大。在这里，我们评估了单荧光蛋白报道分子，双荧光蛋白报道分子的定量性能，以及响应酵母中TAG（琥珀色）密码子后ncAA插入的细胞表面展示蛋白报道分子。尽管不同的报道分子支持不同水平的表观通读效率，但基于流式细胞仪的双报道分子评估可以得到在所有评估条件下均显示出一致的定量趋势和精确度的测量结果。对双荧光蛋白报告基因体系的进一步研究表明，定量输出受终止密码子位置以及N和C端荧光蛋白同一性的影响。双荧光蛋白报告基因和酵母的“即插即用”版本均支持对几种单基因敲除菌株中ncAA掺入的定量分析，揭示了可增强ncAA掺入效率且不影响保真度的菌株。我们的研究揭示了有关酵母中报道基因系统性能以及如何有效部署此类报道基因的关键细节。这些发现对如何设计ncAA掺入系统和蛋白质翻译设备，以更好地适应替代性遗传密码，以扩大生物合成蛋白质的化学多样性具有重大意义。