Expanding the Genetic Code of a Synthetic Yeast

形成自然界化学工具箱的二十种典型氨基酸建立并限制了细胞内基于蛋白质的化学相互作用的可能性。将这些构建模块扩展到包含具有外来侧链的非规范氨基酸（ncAAs），为生物设计打开了一个非凡的空间——从蛋白质-蛋白质相互作用的研究到设计具有新生物功能的酶，对整个合成生物学产生了影响。虽然遗传密码扩展的努力主要集中在大肠杆菌上，但合成酵母Sc2.0的诞生，使得真核生物基因组的扩展得以巩固，其中琥珀色终止密码子已经被基因组重构释放出来进行扩展。然而，促进野生型ncAA在Sc2.0中的整合将是一项多方面的任务，需要对转化途径中的多个步骤进行重新设计和优化。即：工程真核延伸因子1 α (eEF1A)促进正交酰基化tRNA的识别，以改善tRNA -核糖体的相互作用；内源性真核释放因子1 （eRF1）的工程设计以防止琥珀色密码子的竞争性抑制；高效、正交tRNA-氨基酰tRNA合成酶对的工程设计；以及新的核糖体的工程设计，以适应新的ncaa侧链。由于转译机器的每个齿轮都会影响其其余部件的效率，因此每个元件的处理顺序可能会影响整个系统的优化能力，而上游机器是初始探索的主要重点。在本博士研究中，希望初步建立一组正交tRNA-氨基酰基tRNA合成酶对，以基准机械工程改进的进展，并为酵母中ncAA的掺入提供一些初始功能。同时，建立一个没有eRF1琥珀色密码子竞争的背景菌株将是促进进一步改进的关键因素。在此之后，我们预计将在提高正交氨基酰基tRNA合成酶- tRNA对的效率以及利用Sc2.0中允许基因组随机化的独特SCRaMbLE功能优化tRNA对的表达方面做出相当大的努力。

The twenty canonical amino acids that form natures chemical toolbox establish and confine the possibilities of protein-based chemical interactions within the cell. Expanding these building blocks to encompass non-canonical amino acids (ncAAs) with exotic side chains, unlocks an extraordinary space for biological design - having impacts across synthetic biology, from the study of protein-protein interactions, to designing enzymes with new biological functions. Although efforts in genetic code expansion have primarily focused in Escherichia coli, the genesis of the synthetic yeast, Sc2.0, permits consolidating efforts into expansion of the eukaryotic genome where the amber stop codon has been liberated for expansion by genomic refactoring. However, facilitating wild-type levels of ncAA incorporation in Sc2.0 will be a multi-faceted task, requiring re-engineering and optimisation of multiple steps in the translational pathway. Namely: the engineering of the eukaryotic elongation factor 1 alpha (eEF1A) to promote recognition of orthogonal acylated tRNAs to improve tRNA - ribosome interaction; the engineering of the endogenous eukaryotic release factor 1 (eRF1) to prevent competitive suppression of amber codons; the engineering of highly efficient, orthogonal tRNA-aminoacyl tRNA synthetase pairs; and the engineering of new ribosomes to accommodate the novel side chains of ncAAs. As each cog of the translational machinery will impact the efficiency of its remaining parts, the order in which each element is addressed will likely impact the ability to optimise the overall system, with upstream machinery being the primary focus in initial exploration. Within this doctoral investigation, it is hoped to initially establish a set of orthogonal tRNA-aminoacyl tRNA synthetase pairs to benchmark the progress of improvement as machinery is engineered as well as providing some initial functionality for ncAA incorporation in yeast. In parallel, establishing a background strain without eRF1 amber codon competition will be a critical element in facilitating further improvements. After which, it is envisioned that considerable efforts will be made into improving the efficiency of the orthogonal aminoacyl tRNA synthetase - tRNA pair and the optimisation of the pairs expression by use of the unique SCRaMbLE function available to Sc2.0 that allows for genome randomisation.