Biochemistry of bacterial dissimilatory metal reduction

异化金属还原是一个呼吸过程，在各种细菌和古生菌中运作。这些微生物能够使用氧化金属，如Fe(III), Mn(IV), Cr（VI）或U（VI）作为其厌氧电子传递链的终端电子受体。希瓦氏菌是研究这一过程的两种模式生物之一。然而，即使经过几十年对异化金属还原的研究，电子向金属电子受体传递所需的最小蛋白质集仍然是未知的。我正在寻求一种基于合成生物学的新方法来解决这个问题。因此，本研究的目的是通过表达同源葡萄球菌的潜在关键基因，将大肠杆菌转化为一种异化的金属减速器。利用这种技术，我们已经建立了一种能够以螯合铁（III）作为终端电子受体进行呼吸的菌株，从而表明了该方法的适用性。通过插入更多的基因，为可能的电子传递链化合物，我们想进入下一个阶段，即建立电子转移到晶体铁和锰的形式。我们将使用几种平行的方法来检测铁和锰还原链的潜在关键参与者，包括质谱，纯化和后续表征以及靶向基因缺失。对铁还原链进行了一系列的原理验证实验。这些体外实验揭示了蛋白质间的电子转移反应，并在体内实验中得到了验证。所设计的大肠杆菌菌株有望进一步用于解决蛋白质定位和复合物形成问题，并作为进一步结晶研究目标的异系表达平台。

Dissimilatory metal reduction is a respiratory process that operates in various Bacteria and Archaea. These microorganisms are able to use oxidized metals like Fe(III), Mn(IV), Cr(VI), or U(VI) as terminal electron acceptors of their anaerobic electron transport chains. Shewanella oneidensis is one of the two model organisms to study this process. However, even after decades of research on dissimilatory metal reduction, the essential minimal protein set for the electron transport to metallic electron acceptors is still unknown. I am pursuing a novel synthetic biology based approach to tackle this problem. Therefore, the goal of the proposed study is to convert Escherichia coli into a dissimilatory metal reducer through expression of potential key genes from S. oneidensis. Using this technique we have already established a strain that is able to respire with chelated Fe(III) as terminal electron acceptor and have thereby shown the applicability of the method. Via the insertion of further genes for possible compounds of the electron transport chain from S. oneidensis we want to proceed to the next level, being to establish an electron transfer to crystalline iron and manganese forms. We will detect potential key players for the reduction chain to ferric iron and manganese using several parallel approaches, which include mass spectrometry, purification and following characterization as well as targeted gene deletions. A series of proof of principle experiments for the reduction chain to ferric iron were already conducted. These in vitro experiments revealed interprotein electron transfer reactions and were also verified by in vivo experiments. The designed E. coli strains are supposed to be further used to address questions of protein localization and complex formation and as a platform for heterologues expression of targets for further crystallization studies.