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

本计画旨在设计、建立数学模型并在E. coli是一种新型的合成资源回收系统，（a）自动释放被mRNA-核糖体“不间断”复合物浪费性地隔离的核糖体，其通常由合成生物学蛋白质的过表达产生（B）加速由不间断复合物产生的误译蛋白质的降解，从而使被这些误译蛋白质隔离的氨基酸再循环（c）改善宿主细胞适应性，从而改善生长和蛋白质生产速率，通过自动增加对合成DNA过度表达的蛋白质的降解速率来应对负担。我们的合成资源回收系统依赖于模块化的再循环，工程化的核糖体救援机制，自然使用的细菌细胞，以检测和减轻浪费隔离的两个最重要的细胞资源，即核糖体和氨基酸。为此，我们将重新设计主要的核糖体拯救机制，即tmRNA介导的转译系统，以创建一个模块化系统，该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，mCherry）融合。通过该系统，误译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拯救系统将使我们能够轻松地定量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电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上停滞和形成不停止复合物的主要原因是否是缺乏带电荷的tRNA（由于游离氨基酸的严重消耗）以及这是否可以通过增加在过表达或错误折叠的蛋白质中螯合的氨基酸的再循环速率来缓解，从而减少“不停”的mRNA-核糖体复合物的形成。这一研究的另一个令人兴奋的方向是，了解如何控制核糖体拯救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劣势的系统。这可以用作合成生物学的一种新的生物安全机制，因为细胞可以被设计成可预测地表现低于其天然对应物的水平，从而提供控制细菌定殖能力的直接手段。因此，建设一个可控制的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的国家。我们将在创造“更适合用途”的工程细胞方面迈出关键的一步，为它们配备一个可控的资源回收系统，增加宿主细胞适应性，同时保持合成生物学功能和蛋白质生产产率，（B）通过控制核糖体拯救机制提高生物安全性，这对于宿主细胞生存力是必需的，（c）提高合成生物学构建体在相同或甚至不同细菌宿主的不同菌株之间的可靠性和可移植性，以及（d）更深入地了解资源分配及其如何影响东道国适应性和生产力。

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.