Collaborative Research: Unravelling mechanisms of Fe oxidation using syntheic biology and biochemistry

铁氧化细菌（FeOB）利用地球丰富的铁来生长。它们形成氧化铁或铁锈，隔离有毒金属和营养物质。通过了解这些微生物如何在环境中利用铁，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。这项研究将发展在自然和工程系统中监测铁氧化的能力，并进一步优化使用原位或工程生物的过程。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，培养科学的创造性技能，并帮助他们准备好利用微生物学来应对环境和生物技术挑战。外展活动包括为即将进入大学的第一代中学女生举办的“让微生物为我们工作！”研讨会，以及一系列公开讲座、市政厅、视频和播客。最重要的信息是，微生物是生态系统的有用组成部分，可以用来满足环境和社会需求。本项目主要研究两种可能的铁氧化酶系统，即中性粒细胞FeOB的外膜细胞色素-孔蛋白Cyc2和MtoAB。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所需的信息。因此，该项目的目的是证明和表征Fe（II）氧化纯化Cyc2和MtoAB；表达铁氧化酶Cyc2和MtoAB的工程希瓦氏菌的底盘应变比较和优化功能的环境相关菌株；并利用合成生物学开发嗜中性自养FeOB的遗传工具。铁氧化酶Cyc2和MtoAB序列将取自一种常见的淡水FeOB， Sideroxydans lithotrophicus ES-1，异源表达，并在一系列环境相关条件下表征氧化还原电位和活性。此外，一种新的底盘生物，希瓦氏菌，将被设计表达Cyc2和MtoAB，然后在各种条件下测定铁氧化活性。同时，该项目还将开发工具和方法来驯化FeOB。这些遗传系统的发展将使这个和其他研究小组能够查询和验证其他难以抗拒的途径。这项工作将进一步证实Cyc2和MtoAB的铁氧化酶功能，并为其生化机制和功能优化条件提供新的思路。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。

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.