Abstract：
Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyrCUA (Ec. tRNATyrCUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans, they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O-methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function.
摘要：
遗传密码扩展技术允许将非天然氨基酸（UAA）掺入蛋白质中，这对蛋白质工程很有用，合成生物学,和基因治疗。尽管其在各种物种中的潜在应用，丝状真菌仍未被探索。本研究旨在通过在构巢曲霉中开发这些技术来解决这一差距。我们将琥珀终止密码子引入了在A.nidulans中表达的报告基因内的特定序列中，并用CUA替换了真菌tRNATyr的反密码子。这导致了靶蛋白的合成，确认真菌中琥珀抑制的发生。当外源大肠杆菌tRNATyrCUA(Ec。tRNATyrCUA）和大肠杆菌酪氨酰-tRNA（Ec。TyrRS）被引入纳杜兰，他们通过琥珀抑制成功合成了靶蛋白，并被证明与真菌翻译系统正交。通过替换野生型Ec。具有对UAAO-甲基-L-酪氨酸具有更高亲和力的突变体的TyrRS，真菌系统能够启动UAA标记蛋白（UAA蛋白）的合成。我们通过几个合理的修饰进一步增加了UAA蛋白的表达水平。一种遗传密码扩展技术的成功开发，为研究真菌蛋白质的结构和功能提供了一种潜在的有价值的方法。
