Collaborative research: Unravelling mechanisms of Fe oxidation using synthetic biology and biochemistry

氧化铁细菌(FeOB)利用地球上丰富的铁来生长。它们形成铁氧化物或铁锈，隔离有毒金属和营养物质。通过了解这些微生物如何利用环境中的铁并开发驯化它们的工具，就有可能利用这种力量对水、废水和沉积物进行生物修复。这项研究将发展在天然和工程系统中监测铁氧化的能力，并进一步使用原位或工程生物优化过程。该项目将在微生物学、生物化学和合成生物学方面教育不同的人群，发展科学创造技能，并帮助他们准备使用微生物学来应对环境和生物技术挑战。外展活动包括“让微生物为我们工作！”为中学女生举办的讲习班，她们是第一代大学毕业生，以及一系列公开讲座、市政厅、视频和播客。最重要的信息是，微生物是生态系统有用的组成部分，可以利用它们来满足环境和社会需求。本项目的重点是两个可能的铁(Fe)氧化酶系统，外膜细胞色素-孔蛋白Cyc2和MtoAB，来自中性FeOB。目的是验证铁氧化活性并确定功能差异，为在合成系统中应用这些铁氧化酶提供必要的信息。因此，本项目的目标是展示和表征纯化的Cyc2和MtoAB氧化Fe(II)；在工程菌株中表达铁氧化酶Cyc2和MtoAB，以比较和优化与环境相关的菌株的功能；并利用合成生物学开发中性自养FeOB的基因工具。铁氧化酶Cyc2和MtoAB序列将取自一种常见的淡水FeOB，Sideroxdans Rock otriicus ES-1，异源表达，并在一系列与环境相关的条件下表征氧化还原电位和活性。此外，还将设计一种新的底盘生物，白氏希瓦氏菌，表达Cyc2和MtoAB，并在不同条件下测定其铁氧化活性。同时，该项目还将开发驯化FeOB的工具和方法。这些基因系统的开发将使这个研究小组和其他研究小组能够查询和验证其他顽固的途径。这项工作将证实Cyc2和MtoAB的铁氧化酶功能，并提供对生化机制和功能的最佳条件的洞察。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Iron-oxidizing bacteria (FeOB) take advantage of Earth's abundant iron for growth. They form iron oxides or rust that sequester toxic metals and nutrients. By understanding how these microbes use iron in the environment and developing tools to domesticate them, it would be possible to harness this power for bioremediation of water, wastewater, and sediment. This research would develop the ability to monitor iron oxidation in natural and engineered systems, and furthermore optimize the process using in situ or engineered organisms. This project will educate a diverse population in microbiology, biochemistry, and synthetic biology, to develop scientific creative skills and help prepare them to use microbiology to address environmental and biotechnical challenges. Outreach activities include a "Making microbes work for us!" workshop for girls in middle school, who are first-generation college-bound students, and a range of public lectures, town halls, videos, and podcasts. The overarching message is that microbes are useful, integral parts of ecosystems, which can be harnessed to meet environmental and societal needs. This project focuses on two putative iron (Fe) oxidase systems, outer membrane cytochrome-porins Cyc2 and MtoAB, from neutrophilic FeOB. The objective is to validate the Fe oxidation activity and determine differences in function, providing information required for applying these Fe oxidases in synthetic systems. Thus, the project aims are to demonstrate and characterize Fe(II) oxidation by purified Cyc2 and MtoAB; express Fe oxidases Cyc2 and MtoAB in an engineered Shewanella oneidensis chassis strain to compare and optimize function in an environmentally-relevant strain; and develop genetic tools in neutrophilic autotrophic FeOB using synthetic biology. The Fe oxidase Cyc2 and MtoAB sequences will be taken from a common freshwater FeOB, Sideroxydans lithotrophicus ES-1, heterologously expressed, and characterized for redox potential and activity under a range of environmentally-relevant conditions. In addition, a novel chassis organism, Shewanella oneidensis, will be engineered to express Cyc2 and MtoAB, then assayed for Fe oxidation activity under various conditions. In parallel, the project will also develop tools and methods to domesticate FeOB. Development of these genetic systems will enable this and other research groups to query and verify otherwise recalcitrant pathways. This work will substantiate Fe oxidase function of Cyc2 and MtoAB, and provide insight into the biochemical mechanisms and the optimal conditions of function.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.