Cui Zhenling, Johnston Wayne A, Alexandrov Kirill
Synthetic Biology Laboratory, School of Biology and Environmental Science, Queensland University of Technology, Brisbane, QLD, Australia.
Front Bioeng Biotechnol. 2020 Sep 28;8:1031. doi: 10.3389/fbioe.2020.01031. eCollection 2020.
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 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 -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 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 but are equally applicable to the 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 strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.
合成生物学有望通过拓展天然化学空间之外的大分子多样性，给生命科学和生物医学带来变革。通过密码子重新分配使用非标准氨基酸（ncAA）已在蛋白质结构和相互作用分析、翻译后修饰的引入、受限肽的生产、抗体-药物偶联物及新型酶等方面有了多种应用。然而，同时编码多种非标准氨基酸需要复杂的工程操作，且有时会受到细胞对非标准氨基酸摄取能力差的限制。相比之下，无细胞蛋白质合成系统的开放性通过控制翻译组分和试剂的水平及种类，为生物合成机制的操作和重新利用提供了更大的自由度，并允许同时掺入具有非标准侧链甚至主链（N-甲基、D-、β-氨基酸、α-羟基酸等）的多种非标准氨基酸。本综述聚焦于两种最常用的基于无细胞的蛋白质合成系统：基于细胞提取物的系统和基于PURE的系统。前者是一种含有超过500种蛋白质的生物混合物，而后者由38种单独纯化的生物分子组成。我们阐述了这两种系统的组成，并讨论了它们各自的优点和应用。此外，我们剖析了掺入非标准氨基酸所需的翻译组分，并汇编了可通过不同酰化方法掺入多肽的非标准氨基酸列表。我们强调了在使用非天然碱基对增加正交密码子库以及使用tRNA特异性核酶进行酰化方面的最新进展。我们总结了翻译机制（如tRNA、氨酰-tRNA合成酶、延伸因子和核糖体）工程方面的进展，以实现对结构具有挑战性的非标准氨基酸的有效掺入。我们注意到，生物合成机制的许多工程化组分是为使用[原文此处似乎有缺失信息]系统而开发的，但同样适用于[原文此处似乎有缺失信息]系统。这些内容包含在综述中，以便为非标准氨基酸掺入提供全面概述，并为无细胞系统的未来发展提供新见解。最后，我们强调了基因组工程方面令人兴奋的进展，产生了不含琥珀密码子和一些冗余有义密码子的菌株。这些菌株可用于制备提供多种重新分配选项的细胞提取物。
