Genetical approach

Altering the metabolic pathway of fumarate by knocking out the fumarate antiporter DcuB in E. coli and manipulating different fumarate reductases.

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

Figure 1: The electron flow in the respiratory chain.

Fumarate Reductase

We expressed the fumarate reductase (frd) from Escherichia coli under controll of the T7 promotor in different E. coli strains. This enzyme is located in the inner membrane and leads to an increased succinat production in the cell. The fumarate reductase can serve as e key enzyme in electron transfer into the cell and use the reduced mediator as electron donor.
Overview:
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.