Biddle Wil, Schwark David G, Schmitt Margaret A, Fisk John D
Department of Chemistry, University of Colorado Denver, Denver, CO, United States.
Front Chem. 2022 Feb 17;10:815788. doi: 10.3389/fchem.2022.815788. eCollection 2022.
The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters. In sense codon reassignment, the heterogeneous proteins produced greatly complicate the development of screens for variants of the orthogonal machinery with improved activity. We describe the use of a previously-reported fluorescence-based screen for sense codon reassignment as the first step in a directed evolution workflow to improve the incorporation of a ncAA in response to the Arg AGG sense codon. We first screened a library with diversity introduced into both the orthogonal tyrosyl tRNA anticodon loop and the cognate aminoacyl tRNA synthetase (aaRS) anticodon binding domain for variants that improved incorporation of tyrosine in response to the AGG codon. The most efficient variants produced fluorescent proteins at levels indistinguishable from the translation machinery decoding tyrosine codons. Mutations to the aaRS that were found to improve tyrosine incorporation were transplanted onto a aaRS evolved for the incorporation of -azidophenylalanine. Improved ncAA incorporation was evident using fluorescence- and mass-based reporters. The described workflow is generalizable and should enable the rapid tailoring of orthogonal machinery capable of activating diverse ncAAs to any sense codon target. We evaluated the selection based improvements of the orthogonal pair in a host genomically engineered for reduced target codon competition. Using this particular system for evaluation of arginine AGG codon reassignment, however, strains with genomes engineered to remove competing tRNAs did not outperform a standard laboratory strain in sense codon reassignment.
将遗传密码扩展到单一类型的非标准氨基酸（ncAA）之外，受到重新分配有义密码子含义的低效机制的阻碍。利用定向进化提高有义密码子重新分配效率的一个主要障碍是，部分有义密码子重新分配会导致全长蛋白质的异质混合物，其中针对目标密码子掺入的要么是ncAA，要么是天然氨基酸。在终止密码子抑制系统中，掺入失败会导致截短的蛋白质；活性的提高可以从蛋白质产量增加或下游报告基因的产生来推断。在有义密码子重新分配中，产生的异质蛋白质极大地复杂化了用于筛选具有改进活性的正交机制变体的筛选方法的开发。我们描述了使用先前报道的基于荧光的有义密码子重新分配筛选方法，作为定向进化工作流程的第一步，以提高响应精氨酸AGG有义密码子掺入ncAA的效率。我们首先筛选了一个文库，该文库在正交酪氨酰tRNA反密码子环和同源氨酰tRNA合成酶（aaRS）反密码子结合域中都引入了多样性，以寻找能够提高响应AGG密码子掺入酪氨酸效率的变体。最有效的变体产生的荧光蛋白水平与解码酪氨酸密码子的翻译机制无法区分。发现能提高酪氨酸掺入效率的aaRS突变被移植到一个为掺入叠氮苯丙氨酸而进化的aaRS上。使用基于荧光和质量的报告基因可以明显看出ncAA掺入效率得到了提高。所描述的工作流程具有通用性，应该能够快速定制能够将各种ncAA激活到任何有义密码子靶点的正交机制。我们在经过基因组工程改造以减少目标密码子竞争的宿主中评估了正交对基于选择的改进。然而，使用这个特定系统评估精氨酸AGG密码子重新分配时，经过基因组工程改造以去除竞争性tRNA的菌株在有义密码子重新分配方面并不比标准实验室菌株表现更好。
