Directed Evolution of an Orthogonal Quadruplet Codon-based Genetic Code

基于正交四联体密码子的遗传密码的定向进化

• 批准号: EP/Y014154/1
• 负责人: Jason Chin
• 金额: $ 23.84万
• 依托单位: MRC Laboratory of Molecular Biology
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FY014154%2F1
• 关键词: Directed; Evolution; Orthogonal; Quadruplet; Codon

Strategies to expand the genetic code with non-canonical amino acids provide an unparalleled ability to site-specifically endowproteins with new functionalities. This has enabled a multitude of strategies to study protein function, structure, interactions, andlocalisation by, for example, encoding biophysical probes or photocaged residues. Although orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been generated for the incorporation of >150 structurally diverse amino acids, the number of distinct amino acidsthat can be added to the genetic code simultaneously is restricted by a lack of free codons. Recently, the synthesis of an E. coligenome wherein three codons were recoded to synonyms has enabled straightforward access to a 23-amino acid genetic code. Ipropose to drastically expand the utility of this E. coli strain by splitting the three blank triplet codons into twelve quadruplet codonsto be decoded by quadruplet tRNAs. Significantly, this approach eliminates the competition of quadruplet tRNAs with endogenoustRNAs and removes the risk of toxic frameshifting of the proteome. I will establish a directed evolution strategy to evolve highly activeand specific quadruplet tRNAs. Subsequently, I will combine sets of mutually orthogonal aminoacyl-tRNA synthetase/quadruplettRNA pairs in the recoded E. coli strain, establishing an up-to-32-amino acid genetic code. Next, I will utilise this condensedquadruplet codon-based genetic code to evolve an orthogonal ribosome for improved quadruplet but suppressed triplet codondecoding, creating an orthogonal quadruplet codon genetic code. DEQode will thus unlock the potential of quadruplet codondecoding, provide unprecedented capacity to encode multiple non-canonical amino acids into proteins simultaneously, and generatethe first exclusively-quadruplet orthogonal genetic code. This multidisciplinary project will utilise my protein chemistry expertisewhile providing me with extensive synthetic biology training.

用非经典氨基酸扩展遗传密码的策略提供了无与伦比的能力，使蛋白质具有新的功能。这使得许多策略来研究蛋白质的功能，结构，相互作用和定位，例如，编码生物物理探针或光笼残基。虽然正交氨酰-tRNA合成酶/tRNA对已经产生用于掺入>150个结构上不同的氨基酸，但是可以同时添加到遗传密码中的不同氨基酸的数量受到缺乏自由密码子的限制。最近，合成了一个E.其中三个密码子被重新编码为同义词的大肠杆菌基因组使得能够直接获得23个氨基酸的遗传密码。我建议大幅度扩大这种E.将3个空白三联体密码子拆分成12个四联体密码子，再由四联体tRNA进行解码。值得注意的是，这种方法消除了四联体tRNA与内源性tRNA的竞争，并消除了蛋白质组有毒移码的风险。我将建立一个定向进化策略来进化出高活性和特异性的四联体tRNA。随后，我将联合收割机在重新编码的E.大肠杆菌菌株，建立了高达32个氨基酸的遗传密码。接下来，我将利用这种基于四联体密码子的遗传密码来进化一种正交核糖体，以改进四联体密码子解码，但抑制三联体密码子解码，从而创建一种正交四联体密码子遗传密码。因此，DEQode将释放四联体密码子解码的潜力，提供前所未有的能力，将多个非经典氨基酸同时编码到蛋白质中，并产生第一个排他性的四联体正交遗传密码。这个多学科项目将利用我的蛋白质化学专业知识，同时为我提供广泛的合成生物学培训。