A Re-evaluation of Electron-Transfer Mechanisms in Microbial Electrochemistry: Shewanella Releases Iron that Mediates Extracellular Electron Transfer

Exoelectrogenic bacteria can couple their metabolism to extracellular electron acceptors, including macroscopic electrodes, and this has applications in energy production, bioremediation and biosensing. Optimisation of these technologies relies on a detailed molecular understanding of extracellular electron-transfer (EET) mechanisms, and Shewanella oneidensis MR-1 (MR-1) has become a model organism for such fundamental studies. Here, cyclic voltammetry was used to determine the relationship between the surface chemistry of electrodes (modified gold, ITO and carbon electrodes) and the EET mechanism. On ultra-smooth gold electrodes modified with self-assembled monolayers containing carboxylic-acid-terminated thiols, an EET pathway dominates with an oxidative catalytic onset at 0.1V versus SHE. Addition of iron(II)chloride enhances the catalytic current, whereas the siderophore deferoxamine abolishes this signal, leading us to conclude that this pathway proceeds via an iron mediated electron transfer mechanism. The same EET pathway is observed at other electrodes, but the onset potential is dependent on the electrolyte composition and electrode surface chemistry. EET pathways with onset potentials above -0.1V versus SHE have previously been ascribed to direct electron-transfer (DET) mechanisms through the surface exposed decaheme cytochromes (MtrC/OmcA) of MR-1. In light of the results reported here, we propose that the previously identified DET mechanism of MR-1 needs to be re-evaluated

Electrode-assisted acetoin production in a metabolically engineered Escherichia coli strain

Background This paper describes the metabolic engineering of Escherichia coli for the anaerobic fermentation of glucose to acetoin. Acetoin has well-established applications in industrial food production and was suggested to be a platform chemical for a bio-based economy. However, the biotechnological production is often hampered by the simultaneous formation of several end products in the absence of an electron acceptor. Moreover, typical production strains are often potentially pathogenic. The goal of this study was to overcome these limitations by establishing an electrode-assisted fermentation process in E. coli. Here, the surplus of electrons released in the production process is transferred to an electrode as anoxic and non-depletable electron acceptor. Results In a first step, the central metabolism was steered towards the production of pyruvate from glucose by deletion of genes encoding for enzymes of central reactions of the anaerobic carbon metabolism (ΔfrdA-D ΔadhE ΔldhA Δpta–ack). Thereafter, the genes for the acetolactate synthase (alsS) and the acetolactate decarboxylase (alsD) were expressed in this strain from a plasmid. Addition of nitrate as electron acceptor led to an anaerobic acetoin production with a yield of up to 0.9 mol acetoin per mol of glucose consumed (90% of the theoretical maximum). In a second step, the electron acceptor nitrate was replaced by a carbon electrode. This interaction necessitated the further expression of c-type cytochromes from Shewanella oneidensis and the addition of the soluble redox shuttle methylene blue. The interaction with the non-depletable electron acceptor led to an acetoin formation with a yield of 79% of the theoretical maximum (0.79 mol acetoin per mol glucose). Conclusion Electrode-assisted fermentations are a new strategy to produce substances of biotechnological value that are more oxidized than the substrates. Here, we show for the first time a process in which the commonly used chassis strain E. coli was tailored for an electrode-assisted fermentation approach branching off from the central metabolite pyruvate. At this early stage, we see promising results regarding carbon and electron recovery and will use further strain development to increase the anaerobic metabolic turnover rate

Entwicklung bioelektrochemischer Systeme für die Untersuchung von elektrodenabhängigen Konversionsprozessen

In the present dissertation 3 typs of bioelectrochemical systems were developed. The 1st one was a microbial fuel cell which was tested in a domestic sewage treatment plant. An electrical current was produced while organic carbon and N2-compounds were removed from the wastewater. The 2 other systems served for the characterization of electrotrophic microbes. In this context the use of an carbon cathode as the sole electron donor for a genetically modified R. sphaeroides strain was demonstrated

Pre-existing antibodies against polyethylene glycol reduce asparaginase activities on first administration of pegylated <i>E. coli</i> asparaginase in children with acute lymphocytic leukemia

Antibodies against polyethylene glycol (PEG) in healthy subjects raise concerns about the efficacy of pegylated drugs. We evaluated the prevalence of antibodies against PEG among patients with acute lymphoblastic leukemia (ALL) prior to and/or immediately after their first dose of pegylated E.coli asparaginase (PEG-ASNase). Serum samples of 701 children, 673 with primary ALL, 28 with relapsed ALL, and 188 adults with primary ALL were analyzed for anti-PEG IgG and IgM. Measurements in 58 healthy infants served as reference to define cut-points for antibody-positive and -negative samples. Anti-PEG antibodies were detected in ALL patients prior the first PEG-ASNase with a prevalence of 13.9% (anti-PEG IgG) and 29.1% (anti-PEG IgM). After administration of PEG-ASNase the prevalence of anti-PEG antibodies decreased to 4.2% for anti-PEG IgG and to 4.5% for anti-PEG IgM. Pre-existing anti-PEG antibodies did not inhibit PEG-ASNase activity but significantly reduced PEGASNase activity levels in a concentration dependent manner. Although pre-existing anti-PEG antibodies did not boost, pre-existing anti-PEG IgG were significantly associated with firstexposure hypersensitivity reactions (CTCAE grade 2) (p<0.01; Fisher’s exact test). Two of 4 patients with pre-existing anti-PEG IgG and first-exposure hypersensitivity reactions were not switched to Erwinia ASNase and continued on PEG-ASNase with sufficient activities (≥100U/L). Pre-existing anti-PEG antibodies were detected in a considerable proportion of patients with ALL, did not inhibit PEG-ASNase activity but were associated with lower serum PEGASNase activity levels. Patients with pre-existing antibodies may show mild to moderate signs of hypersensitivity reaction after their first PEG-ASNase, which may be successfully addressed by re-challenge

Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes

Shewanella oneidensis is known for its ability to respire on extracellular electron acceptors. The spectrum of these acceptors also includes anode surfaces. Based on this activity, a versatile S. oneidensis based biosensor strain was constructed in which electricity production can be modulated. Construction started with the identification of a usable rate-limiting step of electron transfer to an anode. Thereafter, the sensor strain was genetically engineered to produce a protein complex consisting of the three proteins MtrA, MtrB and MtrF. This complex is associated to the outer membrane and most probably enables membrane spanning electron transfer. MtrF is an outer membrane cytochrome that catalyzes electron transfer reactions on the cell surface. Under anoxic conditions, wild type cells do not express MtrF but rather MtrC as electron transferring outer membrane cytochrome. Still, our analysis revealed that MtrF compared to MtrC overexpression is less toxic to the cell which gives MtrF a superior position for biosensor based applications. Transcription of mtrA, mtrB and mtrF was linked up to an inducible promoter system, which positively reacts to rising l-arabinose concentrations. Anode reduction mediated by this strain was linearly dependent on the arabinose content of the medium. This linear dependency was detectable over a wide range of arabinose concentrations. The l-arabinose biosensor presented in this study proves the principle of an outer membrane complex based sensing method which could be easily modified to different specificities by a simple change of the regulatory elements

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Additional file 3: Table S3. Bacterial strains used in this study

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Additional file 1: Table S1. Oligonucleotides used in this study

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Additional file 5: Table S5. Summary of growth experiments: end products