Shewanella oneidensis MR-1 is a model electroactive bacterium that has been extensively studied for both foundational mechanisms and biotechnology. Despite this, the extent and complexity of the extracellular electron transport (EET) pathway is still being unraveled. This field of study is further complicated because cells modulate the flow of electrons based on rate of flux, redox partner, and whether electrons are being exported or imported into the cell. In this work, I explore the EET pathway of S. oneidensis in the context of microbial electrosynthesis (MES) optimization. MES technology aims to use microorganisms as biocatalysts to drive the formation of useful chemical products in a bioelectrochemical system (BES), ideally using carbon dioxide (CO2) as the substrate. Here, I will examine the native EET systems and synthetic biology efforts to engineer a strain of S. oneidensis capable of such electroautrophy for bioproduction.In Chapter 2, I look at the influence of oxygen (O2) on MES efficiency for 2,3-butanediol generation in S. oneidensis. To do this, butanediol dehydrogenase is expressed in wild-type (WT) S. oneidensis cells to catalyze the NADH-dependent reduction of acetoin to 2,3-butanediol. Our research group previously showed that electron uptake from a cathode to form NADH is an energetically unfavorable reaction and overcame this thermodynamic barrier through expression of the proton pump proteorhodopsin (PR). In the new design, the reaction is coupled to the energetically favorable reduction of O2 by native oxidase; during this bidirectional electron transfer, electrons from the cathode power both reactions. In Chapter 3, I use this same system to reassess the contribution of major cytochrome proteins in the EET pathway during MES. I demonstrate that the outer membrane MtrCAB complex is essential for this process, while other components like CymA and FccA have a more flexible role. Importantly, I show that exogenous flavins are unable to compensate for the loss of natively produced flavins for 2,3-butanediol production, despite their apparent influence on cathodic current. Finally, I reexamine the role of hydrogenases in this process, demonstrating their importance for cell survival on the electrode. Chapters 4 and 5 of this dissertation focus on the use of synthetic biology techniques to install a CO2 fixation pathway in the heterotrophic S. oneidensis. To achieve this goal, I combine in silico metabolic modeling with a CRISPRi knockdown system to create a strain in which multiple substrates are required for biomass synthesis and energy acquisition. By then expressing RuBisCO and PrkA in this strain (∆gpmA pCBB), I then devise a laboratory evolution experiment to generate a strain that will use CO2 to build biomass. In summary, there is still much to be understood about EET in S. oneidensis and an increasing array of bioengineering tools that can be used to this end. This work does just this by exploring the energetics and physiology of S. oneidensis’s EET network, as well as laying the groundwork for a functional electroautotrophic chassis for carbon-neutral bioproduction.
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