Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides.
Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides.
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DOI：
10.3389/fbioe.2020.01031
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发表时间：
2020
影响因子：
5.7
通讯作者：
Alexandrov K
中科院分区：
文献类型：
--
作者：
Cui Z;Johnston WA;Alexandrov K
合成生物学通过在自然化学空间之外扩展大分子多样性，有望彻底改变生命科学和生物医学。通过密码子重配使用非规范氨基酸（ncAAs）在蛋白质结构和相互作用分析、翻译后修饰的引入、约束肽的生产、抗体-药物偶联物和新型酶等方面有多种应用。然而，在体内同时编码多个ncAAs需要复杂的工程设计，有时会受到细胞对ncAAs摄取不良的限制。相比之下，无细胞蛋白质合成系统的开放性通过控制翻译成分和试剂的水平和身份，为操纵和重新利用生物合成机制提供了更大的自由，并允许同时结合具有非规范侧链甚至主干（n-甲基，D-， β-氨基酸，α-羟基酸等）的多个ncaa。本文综述了两种最常用的基于大肠杆菌的无细胞蛋白合成系统；基于细胞提取物和pure的系统。前者是由500个蛋白质组成的生物混合物，而后者由38个单独纯化的生物分子组成。我们描述了这两种体系的组成，并讨论了它们各自的优点和应用。此外，我们剖析了ncAA整合所需的翻译成分，并编制了可以通过不同的酰化方法整合到多肽中的ncAA列表。我们强调了使用非自然核碱基对来增加正交密码子的曲目，以及使用trna特异性核酶进行原位酰化的最新进展。我们总结了翻译机制的工程进展，如trna、氨基酰基trna合成酶、延伸因子和核糖体，以实现结构上具有挑战性的ncaa的有效结合。我们注意到，许多生物合成机械的工程组件是为体内使用而开发的，但同样适用于体外系统。本文对ncAA的合成进行了综述，并为未来无细胞系统的发展提供了新的见解。最后，我们强调了基因组工程的令人兴奋的进展，导致大肠杆菌菌株不含琥珀和一些冗余的意义密码子。这些菌株可用于制备提供多种重新分配选择的细胞提取物。
Synthetic biology holds promise to revolutionize the life sciences and biomedicine via expansion of macromolecular diversity outside the natural chemical space. Use of non-canonical amino acids (ncAAs) via codon reassignment has found diverse applications in protein structure and interaction analysis, introduction of post-translational modifications, production of constrained peptides, antibody-drug conjugates, and novel enzymes. However, simultaneously encoding multiple ncAAs in vivo requires complex engineering and is sometimes restricted by the cell's poor uptake of ncAAs. In contrast the open nature of cell-free protein synthesis systems offers much greater freedom for manipulation and repurposing of the biosynthetic machinery by controlling the level and identity of translational components and reagents, and allows simultaneous incorporation of multiple ncAAs with non-canonical side chains and even backbones (N-methyl, D-, β-amino acids, α-hydroxy acids etc.). This review focuses on the two most used Escherichia coli-based cell-free protein synthesis systems; cell extract- and PURE-based systems. The former is a biological mixture with >500 proteins, while the latter consists of 38 individually purified biomolecules. We delineate compositions of these two systems and discuss their respective advantages and applications. Also, we dissect the translational components required for ncAA incorporation and compile lists of ncAAs that can be incorporated into polypeptides via different acylation approaches. We highlight the recent progress in using unnatural nucleobase pairs to increase the repertoire of orthogonal codons, as well as using tRNA-specific ribozymes for in situ acylation. We summarize advances in engineering of translational machinery such as tRNAs, aminoacyl-tRNA synthetases, elongation factors, and ribosomes to achieve efficient incorporation of structurally challenging ncAAs. We note that, many engineered components of biosynthetic machinery are developed for the use in vivo but are equally applicable to the in vitro systems. These are included in the review to provide a comprehensive overview for ncAA incorporation and offer new insights for the future development in cell-free systems. Finally, we highlight the exciting progress in the genomic engineering, resulting in E. coli strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.