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A novel, fast and efficient resource recycling system for improving the performance of engineered bacteria

一种新颖、快速、高效的资源回收系统，用于提高工程细菌的性能

基本信息

批准号：
EP/P009352/1
负责人：
Guy-Bart Stan
金额：
$ 56.77万
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FP009352%2F1
关键词：
novel fast efficient resource recycling

项目摘要

The proposed project aims to design, mathematically model and experimentally implement in E. coli a novel synthetic resource recycling system that:(a) Automatically releases ribosomes wastefully sequestered by mRNA-ribosomes "non-stop" complexes, which often result from the overexpression of synthetic biology proteins(b) Accelerates the degradation of mistranslated proteins resulting from non-stop complexes and thereby the recycling of the amino acids sequestered by these mistranslated proteins(c) Improves host cell fitness, thereby improving both growth and protein production rates, by automatically increasing in response to burden the degradation rate of proteins overexpressed from synthetic DNA.Our synthetic resource recycling system relies on the modular re-engineering of the ribosome rescue mechanism naturally used by bacterial cells to detect and alleviate the wasteful sequestration of two of their most important cellular resources, i.e. ribosomes and amino acids. To do this we will re-engineer the primary ribosome rescue mechanism, i.e. the tmRNA mediated trans-translational system, to create a modular system that automatically detects mistranslated proteins and adds a variety of tags to them (e.g. mf-Lon, his, HIV-tat tags) or fuses mistranslated proteins with other proteins (e.g. GFP, BFP, mCherry). Through this system, mistranslated proteins can be easily quantified or quickly degraded to efficiently recycle their constituent amino acids.Our novel synthetic ribosome rescue system will allow us to easily quantify the occurrence and impact of non-stop complex formation and amino acid consumption imposed by synthetic biology constructs on their host cells. This will provide the research community with a deeper understanding of the core feedback interactions between synthetic biology circuits and their host cells and of the main mechanisms responsible for these interactions. In particular, this system will allow to test whether the main cause for the stalling of ribosomes on mRNAs and the formation of non-stop complexes is the lack of charged tRNAs (due a severe depletion of free amino acids) and whether this can be alleviated by increasing the recycling rate of amino acids sequestered in overexpressed or misfolded proteins, thereby reducing the formation of "non-stop" mRNA-ribosome complexes.We will demonstrate the gain in fitness and production capacity of E. coli cells equipped with our synthetic resource recycling system and the associated increase in production yields of antibody fragments, which account for nearly 40% of the global biopharmaceutical market.Another exciting direction of this research is that an understanding of how to control ribosome rescue and the re-use of amino acids will also allow us to purposefully design systems that have a controllable fitness disadvantage. This could be used as a novel biosafety mechanism for synthetic biology, as cells could be designed to predictably perform below the level of their natural counterparts, thereby offering a direct means of controlling bacterial colonisation capability. The construction of a controllable resource recycling system could therefore also be beneficial to those seeking to have greater control over genetically modified technologies.In summary, through this project, we will make a crucial step forward in the creation of engineered cells that are "more fit for purpose" by equipping them with a controllable resource recycling system which will (a) increase host cell fitness while maintaining synthetic biology functionality and protein production yield, (b) improve biosafety through the control of the ribosome rescue mechanism, which is essential for host cell viability, (c) improve reliability and portability of synthetic biology constructs across different strains of the same or even different bacterial hosts, and (d) provide a deeper understanding of resource allocation and how it impacts host fitness and productivity.

拟议项目旨在在大肠杆菌中设计、数学建模并实验实施一种新型合成资源回收系统，该系统：（a）自动释放被mRNA-核糖体“不间断”复合物浪费地隔离的核糖体，这通常是由于合成生物学蛋白质的过度表达造成的（b）加速由不间断复合物和产生的错误翻译蛋白质的降解从而回收这些误译蛋白质所隔离的氨基酸(c) 通过自动增加以响应合成 DNA 过度表达的蛋白质的降解率，提高宿主细胞的适应性，从而提高生长和蛋白质生产率。我们的合成资源回收系统依赖于细菌细胞自然使用的核糖体救援机制的模块化重新设计，以检测并减轻两种最重要的细胞资源（即核糖体和氨基酸）的浪费性隔离。为此，我们将重新设计主要核糖体救援机制，即 tmRNA 介导的转译系统，以创建一个模块化系统，自动检测错误翻译的蛋白质并向其添加各种标签（例如 mf-Lon、his、HIV-tat 标签）或将错误翻译的蛋白质与其他蛋白质（例如 GFP、BFP、米切里）。通过这个系统，错误翻译的蛋白质可以很容易地定量或快速降解，以有效地回收其组成氨基酸。我们的新型合成核糖体救援系统将使我们能够轻松地量化合成生物学构建体对其宿主细胞施加的不间断复合物形成和氨基酸消耗的发生和影响。这将使研究界更深入地了解合成生物学回路与其宿主细胞之间的核心反馈相互作用以及负责这些相互作用的主要机制。特别是，该系统将允许测试核糖体在 mRNA 上停滞和形成不间断复合物的主要原因是否是缺乏带电 tRNA（由于游离氨基酸严重耗尽），以及是否可以通过提高隔离在过度表达或错误折叠蛋白质中的氨基酸的回收率来缓解这一问题，从而减少“不间断”复合物的形成 mRNA-核糖体复合物。我们将展示配备我们的合成资源回收系统的大肠杆菌细胞的适应性和生产能力的提高以及抗体片段产量的相关增加，该抗体片段占全球生物制药市场的近40%。这项研究的另一个令人兴奋的方向是，了解如何控制核糖体救援和氨基酸的再利用也将使我们能够有目的地设计具有可控适应性的系统劣势。这可以用作合成生物学的一种新型生物安全机制，因为细胞可以被设计为可预测地低于其天然对应物的水平，从而提供控制细菌定植能力的直接方法。因此，构建可控资源回收系统也可能有利于那些寻求对转基因技术有更大控制权的人。总之，通过这个项目，我们将通过为工程细胞配备可控资源回收系统，在创建“更适合用途”的工程细胞方面迈出关键一步，这将（a）增强宿主细胞的适应性，同时保持合成生物学功能和蛋白质产量，（b）通过以下方式提高生物安全性：核糖体拯救机制的控制对于宿主细胞的生存能力至关重要，(c) 提高合成生物学构建体在相同甚至不同细菌宿主的不同菌株中的可靠性和可移植性，(d) 提供对资源分配及其如何影响宿主适应性和生产力的更深入的了解。