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Recoded organisms engineered to depend on synthetic amino acids

机译：经过重新编码的生物体经过设计可依赖于合成氨基酸

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摘要

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontain-ment 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 receded 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 pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal trans-lational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, 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 harbouring three 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 〜10~(11) cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxo-trophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative 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.
机译：转基因生物（GMO）越来越多地用于研究和工业系统中，以生产高价值的药品，燃料和化学品。遗传隔离和内在生物遏制将提供必要的生物安全措施，以确保这些封闭系统的安全，并使转基因生物在包括生物修复和益生菌在内的开放系统中安全应用。尽管已经设计了通过基本基因调节，可诱导的毒素转换和工程营养缺陷来控制细胞生长的防护措施，但是这些方法由于交叉代谢必需代谢物，必需基因的泄漏表达或遗传突变而受到损害。在这里，我们描述了一系列基因组减退的生物（GRO）的构建，这些生物的生长受到依赖于外源提供的合成氨基酸（sAAs）的多个必需基因表达的限制。我们在产自大肠杆菌的GRO染色体中引入了甲烷甲烷球菌tRNA：氨基酰基-tRNA合成酶对，该染色体缺少所有TAG密码子和释放因子1，赋予该生物以正交的翻译成分将TAG转化为专用的有义密码子适用于sAA。使用多重自动化基因组工程，我们将框内TAG密码子引入22个基本基因中，将其表达与合成苯丙氨酸衍生氨基酸的掺入联系起来。在分离的60种sAA依赖性变体中，一个值得注意的菌株在MurG，DnaA和SerS的保守功能残基中具有三个TAG密码子，并包含靶向的tRNA缺失，可在生物体上培养〜10〜（11）细胞后保持强劲的生长并显示出无法检测的逃逸频率介质中放置7天或在液体介质中放置20天。这是对现有生物遏制方法的重大改进。我们构建了依赖于sAA的合成营养缺陷型，在环境生长测定中不能通过交叉喂养来拯救。这些营养缺陷型GRO具有替代的遗传密码，可通过阻止水平基因转移来赋予遗传隔离，现在它们依赖于合成生物化学构件的使用，从而在工程生物与环境之间推进了正交障碍。

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  • 来源
    《Nature》 |2015年第7537期|89-93|共5页
  • 作者

    Alexis J. Rovner; Adrian D. Haimovich; Spencer R. Katz; Zhe Li; Michael W. Grome; Brandon M. Gassaway; Miriam Amiram; Jaymin R. Patel; Ryan R. Gallagher; Jesse Rinehart; Farren J. Isaacs;

  • 作者单位

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA,Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

    Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA,Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA;

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA,Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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