Furuhata Yuichi, Rix Gordon, Van Deventer James A, Liu Chang C
Department of Biomedical Engineering, University of California, Irvine, CA, USA.
Molecular Biosystems Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Nat Commun. 2025 May 24;16(1):4832. doi: 10.1038/s41467-025-60120-w.
Genetic code expansion (GCE) is a critical approach to the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons (e.g., the amber stop codon). However, evolving new aaRS/tRNA pairs traditionally relies on a labor-intensive process that often yields aaRSs with suboptimal ncAA incorporation efficiencies. Here, we present an OrthoRep-mediated strategy for aaRS evolution, which we demonstrate in 8 independent aaRS evolution campaigns, yielding multiple aaRSs that incorporate an overall range of 13 ncAAs tested. Some evolved systems enable ncAA-dependent translation at single amber codons with similar efficiency as natural translation at sense codons. Additionally, we discover an aaRS that regulated its own expression to enhance ncAA dependency. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms to advance the field of GCE.
遗传密码扩展（GCE）是将非标准氨基酸（ncAA）位点特异性掺入蛋白质的关键方法。GCE的核心是正交氨酰-tRNA合成酶（aaRS）/tRNA对的开发，其中工程化的aaRS识别选定的ncAA并将其加载到解码无义密码子（例如琥珀色终止密码子）的tRNA上。然而，传统上进化新的aaRS/tRNA对依赖于劳动密集型过程，这通常会产生ncAA掺入效率次优的aaRS。在这里，我们提出了一种由OrthoRep介导的aaRS进化策略，我们在8个独立的aaRS进化实验中证明了该策略，产生了多个aaRS，它们掺入了所测试的总共13种ncAA。一些进化系统能够在单个琥珀色密码子处实现ncAA依赖性翻译，其效率与有义密码子处的天然翻译相似。此外，我们发现了一种aaRS，它能调节自身表达以增强ncAA依赖性。这些发现证明了OrthoRep驱动的aaRS进化平台在推进GCE领域方面的潜力。
