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

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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.