Genetical approach

Our first module deals with the construction of an E. coli strain, which is able to accept electrons stimulating its metabolism. We regard on different electron transfer systems: direct and indirect electron transfer.
Direct electron transfer in bacteria is a very complex and not completely cleared up so far. So we focused on the indirect electron transfer via a mediators, which is reduced at the electrode in a electrobiochemical reactor and would be reoxidized again by bacterial cells. In gram-negative bacteria E. coli there are two membranes with a periplasmatic space between, which has to break through in the course of electron transfer.
We worked on three different mediators: neutral red, bromphenol blue and cytochromes. Additionally we are going to construct an electrophilic E. coli strain, which grows by the help of electric power because of increased metabolic activity. (Park et al., 1999)
To realize that, several steps and problems in electron transfer has to be resolved. The electron transfer system consists of different steps. First of all the reduced mediator has to be cross the outer membrane of E. coli cell. For that we are going to use outer membrane porine OprF (BBa_K1172507) provided by iGEM Team Bielefeld-Germany 2013. Crossing the periplasmatic space, the mediator adsorb at inner membrane of E. coli cell. Electrons has to be transferred into cytoplasm, but the mediator should not enter the cell, because regeneration at the electrode is absolutely necessary. So membrane proteins are required to transfer electrons trough the inner membrane. We looked for different oxidoreuctase systems located in bacterial respiration system (shown in figure 1). We focused on respiratory complex II, which contains the succinate dehydrogenase (SDH). This enzyme catalyzes oxidation of succinate into fumarate by transfering electrons to FAD⁺ generating FADH₂. (Iverson et al., 1999; Richardson et al., 1999)
FADH₂ enter the electron transport chain and achieve proton translocation over the inner bacterial membrane. The proton motoric force is used by ATP synthase. Generated ATP effects an increasing metabolic activity. (Gottschalk et al., 1986)
So our aim is to generate a succinate overage in the cytoplasm by increased fumarate reductase activity. The reduced mediator functioned as electron donor for fumarate reductase. Reduction and oxidation between fumarate and succinate creates a loop into the citric acid cycle. In fact electrons are transferred from the mediator to FAD⁺ regenerating ATP via electron transport chain. This concept was successful shown in succinate producing Actinobacillus succinogenes by Park et al., 1999. Naturally E. coli cells release overproduced succinate into the media. This occurs especially under anaerobic conditions because bacteria use fumarate as final electron acceptor instead of oxygen. The resulting succinate would transport out of the cell via C4 carboxylate transporter dcuB. (Janausch, 2001; Unden et al., 1997) In connection to our second module we planed working under oxygen limited condition, hence effective carbon dioxid fixation is possible. Because of that, C4 carboxylate antiporter DcuB has to be knocked out in our E. coli strain.

In conclusion we are going to modify metabolic pathway of fumarate by knockout of C4 carboxylate antiporter DcuB in E. coli and overexpression of different fumarate reductases. Besides outer membrane porine OprF had to be integrated into bacterial chromosome to ensure constitutive expression of OprF and reduce plasmid overload of bacterial cells.

Figure 1: The electron flow in the respiratory chain.

Fumarate Reductase

We worked on expression of different fumarate reductases: Fumarate reductase (Frd) from Escherichia coli and Fumarate reductase (Fum) from Actinobacillus succinogenes both under controll of the T7 promotor in different E. coli strains. In figure 2 our concept is visualized with neutral red as a mediator. Fumarate reductase has a key role in our first module because this enzyme makes sure that electrons are transfered from reduced mediator into bacterial cells. It leads to increased succinate production, which support ATP production and generation of reductive power, for example FADH₂.

Figure 2: The electron flow mediated by redox active mediator in interaction with fumarate reductase in E. coli cell.

Fumarate reductase is part of the anaerobic fumarate respiration in E. coli. The related enzyme in aerobic respiration is succinate dehydrogenase, which catalyse the reaction from succinat to fumarate. The electrons were transferred from succinate to FADH₂ producing fumarate. Succinat dehydrogenase is also a membrane enzyme and it is part of the citric cycle. Fumarate reductase catalyses the reverse reaction of succinate dehydrogenase. Electrons were transferred under anaerobic conditions from FADH₂ to fumarate. Succinate is secreted into the media to take electrons out of the cell. In our project we use fumarate reductase in combination with an extracellular mediator as electron donor to transfer electrons into bacterial cells. The reduced mediator cross the outer membrane of E. coli through outer membrane porine OprF (BBa_K1172507). Mediators diffuse into inner membrane and transfer electrons to fumarate reductase. After that the reduced fumarate reductase transfer electrons to fumarate producing succinate. Succinate can serve as substrate for succinate dehydrogenase, which catalyzes oxidation of succinate into fumarate again. So we create a loop in the citric cycle between fumarate and succinate generating FADH₂ as reductive power in the cell. Electrons are transferred to FAD+, which generate proton translocation from cytosol into the periplasmatic space. The proton motoric force achieve ATP production. So mediator-dependent activity of fumarate reductase serve as energy source for bacterial cells.

C4 Carboxylate Antiporter DcuB

Anaerobic respiration use different alternative electron acceptors, that are less-oxidizing than oxygen. Fumarate can serve as final electron acceptor in anaerobic respiration. Fumarate reductase transfer electrons from electron transport chain to fumarate producing succinate. Succinate leave the cell through C4 carboxylate transporter dcuB. We use fumarate reductase to generate reductive power in bacterial cells. As electron donor we use an extracellular mediator, that should be reduced at the cathode in our electrochemical reactor system. The mediator enter the periplasmatic space through constitutive expressed outer membrane porines (BBa_K1172507). The mediator diffuse in the inner bacterial membrane and get oxidized by fumarate reductase. Fumarate reductase transfer the electrons from the mediator to fumarate. The produced succinate should be regenerated by succinate dehydrogenase into fumarate again. Electrons are transferred to FAD+ and enter bacterial electron transport chain. Naturally E. coli would release succinate into the media under anaerobic conditions. To avoid this, we knocked out the C4 carboxylate antiporter dcuB using Genebridge Red/ET-System. In the same step we integrate the outer membrane porine OprF (BBa_K1172507) into bacterial chromosome under controll of a constitutive promotor (BBa_J23104) This ensure the permeability of outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.

Cytochromes

Figure 1: The electron flow mediated by redox active molecules.

References

• Iverson et al., 1999. Structure of the Escherichia coli Fumarate Reductase Respiratory Complex. Science, vol. 284, pp. 1961-1966
• Janausch, 2001. Rekonstitution des Fumaratsensors DcuS in Liposomen und Transport von Fumarat und Succinat in Escherichia coli. Doctoral dissertation at Johannes Gutenberg-Universität Mainz, Germany
• Gottschalk et al., 1986. Bacterial Metabolism. Springer-Verlag, New York, Berlin, Heidelberg, Tokyo
• Park et al., 1999. Utilization of Electrically Reduced Neutral Red byActinobacillus succinogenes: Physiological Function of Neutral Red in Membrane-Driven Fumarate Reduction and Energy Conservation. Journal of Bacteriology, vol. 181, pp. 2403-2410
• Richardson et al., 1999. Bacterial respiration: a flexible process for a changing environment. Microbiology, vol. 146, pp. 551-571
• Unden et al., 1997. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochimica et Biophysica Acta, vol. 1320, pp. 217-234