A Synthetic Biology Toolbox for Studying Bacterial Transporters

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This collaborative project between the University of Wisconsin-Madison and members of the Department of Energy’s Agile BioFoundry (Oak Ridge and Lawrence Berkeley National labs) investigates bacterial transporters and engineers them to have superior properties for use in biomanufacturing strategies. Biomanufacturing is a means to convert renewable resources into a wide range of chemical products at ambient temperature and pressure with the use of living catalysts. In biomanufacturing, microbial cells operate like processing factories and it is important to have knowledge and control of what enters and exits the cells via transporters. The current transporter knowledgebase contains many gaps including how to export desirable chemical products that are otherwise toxic to cells in high concentrations. Closing these basic science knowledge gaps facilitates the development of superior biomanufacturing microbial hosts and supports the translational research needed to fulfill the scientific mission of the US. Department of Energy. The project contributes to workforce development by providing synthetic biology training to graduate and undergraduate students studying at the intersection of microbiology and engineering. The project includes a partnership with established Research Experience for Undergraduates programs at the University of Wisconsin-Madison to recruit talented underrepresented researchers and mentor them from their summer residence until the end of the bachelor’s career. Through the course of this project, a suite of tools that are broadly useful to the metabolic engineering and biomanufacturing communities, are developed. The goal is to develop and use tools to close knowledgebase gaps surrounding transporters in Gram negative bacteria. The project involves the use of genome wide fitness association assays to perform loss of function studies and to identify putative transporters linked to selectable phenotypes (e.g., uptake of a limiting nutrient, removal of a toxic product). Strains of bacteria for use in complementary gain of function experiments that validate putative transport functions are being developed. Related tools are used to facilitate protein engineering studies that enhance transport function. The research also involves engineering of active exporters and passive permeases with deep saturation mutagenesis techniques and the use of structure-guided and AI-tools to round out a design-build-test-learn campaign. Tools are deposited in public repositories so that the community can easily explore the transporter function of their interest. The research contributes new data to systems biology models such as metabolism reconstructions used for flux balance analysis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.