Genetical approach

Figure 1: Module 1 overview

Our first module deals with the construction of an E. coli strain, which is able to accept electrons stimulating its metabolism. We considered two 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. Because of this we focused on the indirect electron transfer via mediators, which are reduced at the electrode in a electrobiochemical reactor and then reoxidized again inside bacterial cells. In the gram-negative bacteria E. coli there are two membranes with a periplasmatic space between them, which have to be overcome in order to achieve a successful electron transfer.
We worked on three different mediators: neutral red, bromphenol blue and Cytochromes. Additionally we constructed an electrophilic E. coli strain, which grows in the presence of electric current and therefore shows increased metabolic activity. (Park et al., 1999) To realize this aim, several steps and problems in electron transfer have to be resolved. The electron transfer system consists of different steps:
First the reduced mediator has to cross the outer membrane of the E. coli cell. For that we are going to use the outer membrane porine OprF (BBa_K1172507) provided by iGEM Team Bielefeld-Germany 2013. Crossing the periplasmatic space, the mediator adsorbs at the inner membrane of the E. coli cell. Electrons have to be transferred into the 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 oxidoreductase systems located in bacterial respiration system (shown in figure 3).

Figure 2: The electron flow mediated by redox active mediator (for example neutral red) in interaction with fumarate reductase in E. coli cell. The electrochemical reduced mediator has to cross outer membrane via genome integrated outer membrane porines OprF. The fumarate reductase in the inner bacterial membrane get electrons from the mediator for reduction of fumarate into succinate in the cytoplasm. Excretion of succinate is avoid becuase of knocked out C4 carboxylate antiporter DcuB.

We focused on respiratory complex II, which contains the fumarate reductase (Frd) and succinate dehydrogenase (Sdh). Last one catalyzes the oxidation of succinate into fumarate by transferring electrons to FAD⁺ generating FADH₂. (Iverson et al., 1999; Richardson et al., 1999) FADH₂ enter the electron transport chain and achieves 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 functions as an electron donor for the 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 the electron transport chain. This concept was successfully shown in succinate producing Actinobacillus succinogenes by Park et al., 1999. Naturally E. coli cells release overproduced succinate into the medium. This occurs especially under anaerobic conditions because bacteria use fumarate as a final electron acceptor instead of oxygen. Besides this effect could be observed if high fumarate concentrations in the medium are available. The produced succinate would be transported out of the cell via the C4 carboxylate transporter dcuB. (Janausch, 2001; Unden et al., 1997) In connection to our second module we planned on working under oxygen limiting conditions, hence effective carbon dioxid fixation is possible. To achieve an effective electron uptake and prevent any succinate excretion, the C4 carboxylate antiporter DcuB has to be knocked out in our E. coli strain.

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

Figure 3: The electron flow in the respiratory chain. Different complexes of electron transport chain under aerobic conditions are visualized. Free energy (E₀´) change from negative to positive during electron transfer from NADH to oxygen finally.

Fumarate Reductase

We worked on the expression of different fumarate reductases: Fumarate reductase (Frd) from Escherichia coli (BBa_K1465102) and Fumarate reductase (Fum) from Actinobacillus succinogenes (BBa_K1465103; BBa_K1465104 ) both under the control of the T7 promotor in different E. coli strains. In figure 4 our concept is visualized with neutral red as a mediator. The fumarate reductase has a key role in our first module because this enzyme makes sure that electrons are transferred from the reduced mediator into bacterial cells. This leads to an increased succinate production, which support ATP production and generation of reductive power, for example FADH₂.

Figure 4: The electron flow mediated by redox active mediator (for example neutral red) in interaction with fumarate reductase in E. coli cell. The electrochemical reduced mediator has to cross outer membrane via genome integrated outer membrane porines OprF. The fumarate reductase in the inner bacterial membrane get electrons from the mediator for reduction of fumarate into succinate in the cytoplasm. Excretion of succinate is avoid becuase of knocked out C4 carboxylate antiporter DcuB.

Fumarate reductase is part of the anaerobic fumarate respiration in E. coli. It catalyzes the reaction from fumarate into succinate using fumarate as a final electron acceptor in anaerobic fumarate respiration. The related enzyme in aerobic respiration is the succinate dehydrogenase, which catalyses the reaction from succinate to fumarate. The electrons are transferred from succinate to FAD⁺ producing fumarate and FADH₂. The succinate dehydrogenase is also a membrane enzyme and it is part of the citric acid cycle. They both belong to the respiration complex II. Naturally there is no activity of both enzymes at the same time in E. coli cells. (Iverson et al., 1999)
In our project we used the fumarate reductase in combination with an extracellular mediator as electron donor to transfer electrons into bacterial cells. The reduced mediator crosses the outer membrane of E. coli through the outer membrane porine OprF (BBa_K1172507). Mediators adsorb at the inner membrane and transfer electrons to the fumarate reductase. After that the reduced fumarate reductase transfer electrons to fumarate producing succinate. This process has been shown for the fumarate reductase in Actinobacillus succinogenes by Park et al..
Succinate can serve as a substrate for the succinate dehydrogenase, which catalyzes the oxidation of succinate into fumarate again. So we want to create a loop in the citric acid cycle between fumarate and succinate generating FADH₂ as a reductive power in the cell. Electrons are transferred to FAD+, which generate proton translocation from the cytoplasm into the periplasmatic space. The proton motoric force leads to ATP production. To conclude the mediator-dependent activity of the fumarate reductase could serve as an energy source for bacterial cells.
We plan on comparing the activity of the fumarate reductases (Frd) from Escherichia coli KRX and the fumarate reductase (Fum)from Actinobacillus succinogenes working with different mediators, for example neutral red or bromphenol blue, as electron donors.

C4 Carboxylate Antiporter DcuB

Under anaerobic conditions E. coli cells use different alternative electron acceptors instead of oxygen. Partially the bacteria use fumarate respiration, whereby fumarate is reduced into succinate. There are also other potential less-oxidizing substances for bacteria to release their electrons, for example anorganic compounds like nitrate (NO₃^-) or sulfate (SO₄^2-).(Gottschalk et al., 1986) Fumarate respiration leads to succinate excretion through the C4 carboxylate transporter DcuB. It is an antiporter which exchanges fumarate against succinate under anaerobic conditions. Under aerobic condition there is usually no succinate release observed. In connection to the carbon dioxide fixation in our second module we planned on working under oxygen limiting conditions, hence effective carbon dioxid fixation is possible. So in case of oxygen limiting conditions, there could occured partial fumarate respiration in E. coli. Besides there was shown activity of DcuB antiporter in the presence of high fumarate concentrations (Janausch, 2001). To achieve an effective electron uptake and prevent any succinate excretion, the C4 carboxylate antiporter DcuB has to be knocked out in our E. coli strain.
We planned a targeted knockout of the dcuB gene in E. coli KRX using the Genebridge Red/ET-System. In the same step we are going to integrate the outer membrane porine OprF (BBa_K1172507) into the bacterial chromosome under controll of a constitutive promotor (BBa_J23104). This ensure the permeability of the outer membrane and avoid a plasmid overload of the bacteria, because for our system the outer membrane porines are indispensable.

Cytochromes

Besides the intention of using a mediator for indirect electron transfer we additionally focused on the electron transfer via cytochromes. Although the exact mechanism of electron transfer by cytochromes is not clarified up to now there are a few basic approaches that indicate which ones could have an important function. The basic idea of this attempt is to bring a periplasmatic cytochrome which is essential for electron uptake in E. coli and use it as an intermediate electron acceptor. Therefore we decided to work with a key-type cytochrome c, called PccH of Geobacter sulfurreducens because it has been shown that biofilms of this organism are capable of accepting electrons from an electrode. Furthermore it has been demonstrated that the mechanism of electron delivery and electron uptake varies in view of involved proteins. In this case transcriptome analysis by microarray revealed that the corresponding gene ( GSU 3274) of PccH is far more abundant in current-consuming cells using an electrode as electron donor in order to reduce fumarate than the genes known to be essential for electron delivery. Additionally deletion of GSU 3274 inhibits the electron uptake completely and complementation of it afterwards can fully restore electron consumption. Based on this we aim to express PccH (BBa_K1465101) in E. coli to bring the electrons into the cell by reducing the membrane-bound fumarate reductase.(Strycharz et al., 2011; Dantas et al., 2013)
Figure 5 shows the general principle of the electron flow.

Figure 5: The electron flow mediated by redox active molecules.

Therefore the periplasmatic cytochrome leaves the periplasm through the porins OprF (BBa_K1172507) in the outer membrane, gets reduced at the cathode and enters the periplasm again. There the fumarate reductase which is located in the inner phospholipid membrane oxidized the cytochrome again and is thereby the connective element which transfers the electrons into the cell. These supplied electrons increase the succinate production and together with the succinate dehydrogenase the metabolic activity of the cells should be enhanced.
One of the challenges going along with this approach is the correct localization of the c-type-cytochrome in the periplasm. It is documented that PccH has its own signal peptide for localization and therefore we initially want to use this signal peptide(Dantas et al., 2013). Furthermore we consider to use E. coli specific signal peptides afterwards if necessary.
Another challenge is the correct folding of PccH because there are specific cytochrome c maturation (ccm) genes needed. Those genes also exist in E. coli but are normally just expressed under anaerobic conditions (Jensen et al., 2010). In view of module two and three and of the characterization of PccH completely anaerobic conditions are not possible and can not be guaranteed. This is why we have to bring the ccmA-H genes on a plasmid in E. coli to express it under aerobic conditions. Furthermore we bring it under the control of a constitutive anderson promoter by an appropriate design of primer.
The expression strength of both, PccH and ccmA-H, are another challenge because they have to be balanced correctly (Jensen et al., 2010). Therefore we want to express ccmA-H constitutivley whereas the promoter of PccH should be changed to test the optimal ratio between both.

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
• Stycharz, S. M., Glaven, R. H., Coppi, M. V., Gannon, S. M., Perpetua, L. A., Liu, A., Nevin, K. P. &amp. Lovley, D. R. (2011):Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens. In: Bioelectrochemistry 80, pp. 142 - 150.
• Dantas, J. M., Tomaz, D. M., Morgado, L. & Salgueiro, C. A.(2013): Functional charcterization of PccH, a key cytochrome for electron transfer from electrodes to the bacteium Geobacter sulfurreducens. In: FEBS Letters 587, pp. 2662 - 2668.
• Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y., Cohen, B. E., Helms, B. A., Weigele, P., Groves, J. T., Ajo-Franklin, C. M. (2010): Engineering of a synthetic electron conduit in living cells. In: PNAS 107, pp. 19213–19218.