Recoded organisms engineered to depend on synthetic amino acids.
Recoded organisms engineered to depend on synthetic amino acids.
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DOI：
10.1038/nature14095
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发表时间：
2015-02-05
期刊：
影响因子：
64.8
通讯作者：
Isaacs FJ
中科院分区：
文献类型：
--
作者：
Rovner AJ;Haimovich AD;Katz SR;Li Z;Grome MW;Gassaway BM;Amiram M;Patel JR;Gallagher RR;Rinehart J;Isaacs FJ
转基因生物 (GMO) 越来越多地用于研究和工业系统，以生产高价值的药品、燃料和化学品。基因隔离和内在生物防护将提供必要的生物安全措施，以确保这些封闭系统的安全，并使转基因生物能够在开放系统中安全应用，包括生物修复和益生菌。尽管已设计了保护措施来通过必需基因调控、诱导毒素开关和工程营养缺陷型来控制细胞生长，但这些方法会因必需代谢物的交叉喂养、必需基因的表达泄漏或基因突变而受到损害。在这里，我们描述了一系列基因组重新编码生物（GRO）的构建，其生长受到依赖于外源提供的合成氨基酸（sAA）的多种必需基因的表达的限制。我们将詹氏甲烷球菌 tRNA:氨酰基-tRNA 合成酶 (aaRS) 对引入到缺乏所有 TAG 密码子和释放因子 1 的 GRO 染色体中，赋予该生物体正交翻译组件，将 TAG 转化为 sAA 的专用有义密码子。使用多重自动化基因组工程 (MAGE)，我们将框内 TAG 密码子引入 22 个必需基因，将它们的表达与合成的苯丙氨酸衍生氨基酸的掺入联系起来。在分离的 60 个 sAA 依赖性变体中，在 MurG、DnaA 和 SerS 保守功能残基中含有 3 个 TAG 密码子并含有靶向 tRNA 删除的著名菌株在固体培养基上培养 ∼1011 细胞 7 天或在液体培养基中培养 20 天后保持强劲生长，并表现出不可检测的逃逸频率。这是对现有生物防护方法的重大改进。我们构建了依赖于 sAA 的合成营养缺陷型细胞，但在环境生长测定中，这些营养缺陷型细胞并未通过交叉喂养而得以挽救。这些营养缺陷型 GRO 拥有替代遗传密码，通过阻碍水平基因转移来实现遗传隔离，现在依赖于合成生化构件的使用，从而推进工程生物体与环境之间的正交屏障。
Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches, and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here, we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase (aaRS) pair into the chromosome of a GRO that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering (MAGE), we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harboring 3 TAG codons in conserved functional residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for seven days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternate genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.