Microbial electrosynthesis (MES) is an emerging technology which, in the most extreme cases, uses an electroautotrophic biocatalyst to convert CO2 and clean electricity into value-added products. Technologies that combine CO2 remediation efforts with chemical production from fossil-fuel alternatives are promising contributors to the fight against climate change. MES is limited by presently available biocatalysts, which have small product scopes and few genetic engineering tools to improve them. For MES to be an industrially competitive technology, a robust biocatalyst which can be engineered to have a large product scope with high titers must be implemented. This dissertation describes progress toward the development of Shewanella oneidensis and Eschericahia coli as MES hosts.MES uses clean electricity to power the unfavorable reduction reactions that take place in carbon fixation and conversion to fuels and chemicals. Thus, an important aspect of a MES biocatalyst is electroactivity. S. oneidensis is a metal-reducing bacteria that can respire extracellular metals and electrodes. It has a well-characterized extracellular electron transfer pathway, the Mtr pathway, and established genetic engineering tools which make it an attractive candidate for MES. However, S. oneidensis cannot fix CO2. CO2-derived formate assimilation is considered for a CO2-fixing S. oneidensis strain. Formatotrophy would need to be engineered; however, formate is toxic to this organism. As a first step toward engineering formatotrophy in S. oneidensis, several formate tolerant strains were evolved and mutations were characterized (Chapter 2). To drive formate assimilation pathways, low potential electron carriers, such as reduced ferredoxin will be required. Therefore, we characterized the Rnf complex in S. oneidensis, a potential source of low potential electrons, which led to the discovery that Rnf is essential for iron-sulfur cluster biosynthesis and repair in this organism (Chapter 3). This work confirmed that the Rnf complex is a source of reduced ferredoxins in S. oneidensis and could serve as a power source for carbon fixation pathways in the future. In the course of our study of the S. oneidensis Rnf complex, the unusual observation that isopropyl β-D-1-thiogalactopyranoside (IPTG) altered growth of the organism was made. IPTG is often used to induce gene expression in engineered systems and is assumed to have no off-target effects on metabolism or regulation. However, in this work IPTG was found to improve growth of S. oneidensis cultivated with sugar substrate N-acetylglucosamine (Chapter 4). As the use of IPTG is so widespread, researchers in this field should be mindful of our results so false positive correlations can be avoided. Another interesting candidate for a MES biocatalyst is Escherichia coli, one of the most well-studied microorganisms that has been engineered for fermentative biosynthesis of a vast array of compounds. The ability to connect these reactions to CO2 fixation would offer viable means of production of such compounds from a fossil-fuel alternative while helping prevent release of CO2. E. coli requires both the engineering of electroactivity and CO2 fixation to be considered a viable option for MES, as the organism does not natively have either capability. In this work, the biotechnological potential of an electroactive strain of E. coli expressing the Mtr pathway from S. oneidensis is explored (Chapter 5). Overall, this dissertation describes four experimental studies which contribute to overcoming the technical barriers of using S. oneidensis and E. coli as MES biocatalysts.
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