Expanding the Genetic Code of a Synthetic Yeast

形成自然化学工具箱的20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。将这些构建块扩展到包含具有奇异侧链的非规范氨基酸(NCAA)，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，对合成生物学产生了影响。尽管基因密码扩展的努力主要集中在大肠杆菌中，但合成酵母Sc2.0的起源允许将努力整合到真核基因组的扩展中，其中琥珀终止密码子已通过基因组重构释放出来进行扩展。然而，促进野生型水平的NCAA在Sc2.0中的整合将是一项多方面的任务，需要重新设计和优化翻译途径中的多个步骤。即：设计真核延长因子1α(EEF1a)以促进正交酰化tRNAs的识别以改善tRNA与核糖体的相互作用；设计内源真核释放因子1(ERF1)以防止琥珀密码子的竞争性抑制；设计高效、正交的tRNA-氨基酰基tRNA合成酶对；以及设计新的核糖体以适应NCAAs的新侧链。由于转换机械的每个齿轮会影响其剩余部件的效率，因此每个元素的处理顺序可能会影响优化整个系统的能力，上游机械是初始勘探的主要重点。在这项博士研究中，希望初步建立一组正交的tRNA-氨基酰基tRNA合成酶对，以作为机械工程改进进展的基准，并为NCAA在酵母中的掺入提供一些初步功能。同时，在没有eRF1琥珀密码子竞争的情况下建立背景菌株将是促进进一步改进的关键因素。在此之后，预计将做出相当大的努力来提高正交氨酰tRNA合成酶-tRNA对的效率，并通过使用允许基因组随机化的Sc2.0可用的独特的扰乱功能来优化对的表达。

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.