A Synthetic Biology Toolbox for Studying Bacterial Transporters

威斯康辛大学麦迪逊分校和能源部敏捷生物研究中心（橡树岭和劳伦斯伯克利国家实验室）的成员合作开展了这个项目，研究细菌转运体，并通过工程设计使它们具有用于生物制造策略的优越性能。生物制造是利用活性催化剂，在常温常压下将可再生资源转化为多种化学产品的一种手段。在生物制造中，微生物细胞像加工厂一样运作，了解和控制通过转运体进入和离开细胞的物质是很重要的。目前的转运体知识库存在许多空白，包括如何出口理想的化学产品，否则在高浓度下对细胞有毒。缩小这些基础科学知识差距有助于开发卓越的生物制造微生物宿主，并支持完成美国科学使命所需的转化研究。能源部。该项目通过为微生物学和工程学交叉专业的研究生和本科生提供合成生物学培训，促进了劳动力的发展。该项目包括与威斯康星大学麦迪逊分校已建立的本科生研究经验项目的合作伙伴关系，以招募有才华的研究人员，并从他们的夏季住所指导他们，直到学士生涯结束。通过这个项目的过程，开发了一套对代谢工程和生物制造社区广泛有用的工具。目标是开发和使用工具来关闭革兰氏阴性细菌转运体周围的知识库空白。该项目涉及使用全基因组适应度关联分析来进行功能丧失研究，并确定与可选择表型（例如，摄取限制性营养物质，去除有毒产物）相关的推定转运蛋白。正在开发用于功能互补增益实验的细菌菌株，以验证假定的传输功能。相关工具用于促进增强转运功能的蛋白质工程研究。该研究还涉及利用深度饱和诱变技术对主动出口和被动渗透进行工程设计，并使用结构引导和人工智能工具来完成设计-构建-测试-学习活动。工具存放在公共存储库中，以便社区可以轻松地探索他们感兴趣的传输功能。该研究为系统生物学模型提供了新的数据，如用于通量平衡分析的代谢重建。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.