Directed Evolution of an Orthogonal Quadruplet Codon-based Genetic Code

用非规范氨基酸扩展遗传密码的策略提供了一种无与伦比的能力，可以特异性地赋予蛋白质新的功能。这使得研究蛋白质功能、结构、相互作用和定位的多种策略成为可能，例如，通过编码生物物理探针或光笼化残基。虽然正交的氨基酰基-tRNA合成酶/tRNA对已经产生了150个结构不同的氨基酸，但由于缺乏自由密码子，可以同时添加到遗传密码中的不同氨基酸的数量受到限制。最近，一个大肠杆菌基因组的合成，其中三个密码子被重新编码为同义词，使得直接访问一个23个氨基酸的遗传密码成为可能。我建议将这3个空白的三联体密码子分裂成12个四联体密码子，然后用四联体trna解码，从而极大地扩大这一大肠杆菌菌株的效用。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，并消除了蛋白质组毒性移框的风险。我将建立一个定向进化策略来进化高度活跃和特异性的四联体trna。随后，我将在编码的大肠杆菌菌株中组合相互正交的氨基酰基- trna合成酶/四plettrna对，建立一个32个氨基酸的遗传密码。接下来，我将利用这个基于四联体密码子的压缩遗传密码来进化一个正交核糖体，以改进四联体但抑制三联体密码子解码，创建一个正交四联体密码子遗传密码。因此，DEQode将解锁四联体密码解码的潜力，提供前所未有的能力，将多个非规范氨基酸同时编码为蛋白质，并产生第一个完全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。

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.