Costello Alan, Peterson Alexander A, Lanster David L, Li Zhiyi, Carver Gavriela D, Badran Ahmed H
Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
Nat Biotechnol. 2024 Sep 11. doi: 10.1038/s41587-024-02385-y.
Supplementing translation with noncanonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventional Escherichia coli strains with native ribosomes, we develop a plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allowed us to identify 12 mutually orthogonal transfer RNA (tRNA)-synthetase pairs. Enabled by these findings, we evolved and optimized five tRNA-synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Lastly, we extend these resources to an in vivo biosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Our approach will accelerate innovations in multiplexed genetic code expansion and the discovery of chemically diverse biomolecules.
用非标准氨基酸（ncAAs）补充翻译可以产生具有全新功能的蛋白质序列，但现有的ncAA掺入策略存在效率低和上下文依赖性的问题。我们发现密码子使用情况是使用非天然密码子进行有效遗传密码扩展的一个先前未被认识到的因素。仅依靠具有天然核糖体的传统大肠杆菌菌株，我们开发了一种基于质粒的密码子压缩策略，该策略最大限度地减少了上下文依赖性，并提高了四联体密码子处ncAA的掺入效率。我们证实该策略与所有已知的遗传密码扩展资源兼容，这使我们能够鉴定出12对相互正交的转移RNA（tRNA）-合成酶对。基于这些发现，我们进化并优化了五对tRNA-合成酶对，以在正交四联体密码子处掺入多种ncAAs。最后，我们将这些资源扩展到一个体内生物合成平台，该平台可以轻松创建超过100个带有多达三种独特ncAAs的全新肽大环。我们的方法将加速多重遗传密码扩展的创新以及化学多样生物分子的发现。
