Team:TU Delft-Leiden/Project/Life science/EET/CM ET

Carbon Metabolism and Electron Transport

Contemplated in this section is a general description of carbon metabolism of E. coli centered on generation of extracellular electron transport. Furthermore, several intriguing challenges as well as possible consequences with respect to these challenges are indicated.

Shewanella oneidensis natively hosts extracellular electron transfer pathway(s)

Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to utilize insoluble substrates for respiration purposes is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. For a detailed description of characteristics featuring in transfer, see [section].

Respiration in model organism Escherichia coli

Generation of extracellular electron transport (EET) by implementing and expressing genes of S. oneidensis in model organism Escherichia coli is directly influenced by the native carbon metabolism and poses, amongst others, the following questions; which carbon sources can be used as electron donors? Are there possibilities to promote growth of E.coli and make possible EET in one go?

Effectively, necessary for growth are ATP, (carbon) sources of elements essential to growth and proliferation as well as compounds that serve as electron donors and acceptors, and, in anticipation of challenges within the module electron transport, balance in the native quinone / quinol pool as well as NAD(P) / NAD(P)H pools, referred to as the redox balance.

E.coli is quite the versatile model organism in its use of a broad scope of carbon sources and, depending on the source, might grow aerobically or anaerobically. Carbon flux thus strongly depends on growth conditions. On glucose and under aerobic conditions, glycolysis followed by runs of TCA will be the main pathway for generation of ATP. Anaerobic growth on glucose will lead to fermentation into, amongst others, acetate and ethanol.

Native electron transport chain(s) in E.coli

The electron transport chain in a range of organisms, including E. coli, comprises an enzymatic series of electron donors and acceptors in membrane-bound complexes situated in the mitochondrial inner membrane. Each electron donor passes electrons to a more electronegative acceptor, which in turn donates these electrons to another acceptor, a process that continues down the series until, in aerobic cultures, electrons are passed to oxygen, the most electronegative and terminal electron acceptor in the chain. Passage of electrons between donor and acceptor releases energy, which is used to generate a proton gradient across the mitochondrial membrane by actively pumping protons into the intermembrane space, producing a thermodynamic state that has the potential to do work. This process is termed oxidative phosphorylation: ADP is phosphorylated to ATP using the energy of hydrogen oxidation in consecutive steps. A percentage of electrons do not complete the series and directly leak to oxygen.

Bacteria can use a number of different electron donors, dehydrogenases, oxidases and reductases, and several different electron acceptors resulting in multiple electron transport chains operating simultaneously.

First thoughts on this matter included the possible physical separation of the processes oxidative phosphorylation and extracellular electron transport and the saturation of the routes. Are changes in relative flows able to be made, and if so, what is the impact on growth for one and EET on another level? Feasible option with which we continued was anaerobic growth in order to avoid oxygen pulling electrons through the native glycolysis followed by TCA and processed via oxidative phosphorylation.

Relevant basics of carbon metabolism

As mentioned in previous sections, native electron transport chains are present in Escherichia coli and form an interesting sidetrack regarding electron transfer. On a different level, implementing extracellular electron transport in model organism E. coli calls for a conditional electron acceptor; electron transfer should be possible, however, the potential should be such in order for the compound to be able to ‘pull’ and to be reduced. Implementing the system in which growth as well as transfer is made possible is followed by creation of a system or device in which an electrode functions as acceptor and electrons are shuttled out of the cell in order for extracellular electron transfer to take place. Route of choice should, at that point, be the modified pathway of S. oneidensis implemented in E. coli.

Control of operon expression regarding enzymes functional in carbon metabolism is exerted at the transcriptional level in response to the availability of (amongst others) the electron acceptors oxygen, fumarate, and nitrate. Oxygen is the preferred electron acceptor and represses the terminal reductases of anaerobic respiration. Energy conservation is maximal with O2 and lower with, for example, fumarate. By this regulation pathways with high ATP or growth yields are favored. Oxygen, however is (too) strong an acceptor and, theoretically, will not make transport via routes that pose less promising redox potentials possible. Cultivation must thus be anaerobic. Thought experiments included fumarate as an electron acceptor, however, not only the potential is of relevance. Using fumarate, for example, will result in a change in pH. Also, the formation of potentially toxic intermediates will have to be taken into account.

Other aspects that have to be taken into account are the following. If there is a route for E.coli to reduce from a previous accceptor, it will do so and shift metabolism towards the original ‘waste’ product.

Optional routes for generations of ATP and reducing factors have been considered. For example, glucose consumed anaerobically as a source of carbon would be able to generate ATP and necessary cofactors excluding the implemented electron transport system. In general, when looking into carbon source utilization, one would have to prevent alternative routes of generation of ATP. This includes prevention of substrate phosphorylation, resulting in the formation of adenosine triphosphate (ATP) by the direct transfer of a phosphate group to adenosine diphosphate (ADP) from a reactive intermediate. A carbon source is needed in which substrate phosphorylation is not possible. Wij hebben een koolstofbron / systeem nodig waarin substraatfosforylatie niet mogelijk is om te voorkomen dat er op die manier ATP wordt gegenereerd. In cells, it occurs primarily in the cytoplasm (in glycolysis) under both aerobic and anaerobic conditions, must be prevented. Glycerol is a potential source of carbon, however, regarding fluxes, there is unclarity: substrate phosphorylation from glycerol to pyruvate.

Summarizing, in order to choose a feasible source of carbon, it is of importance to check possibilities regarding metabolism where substrate phosphorylation is not an option. Being quite the versatile organism, any fermentation pathway from which the organism is able to achieve a redox balance can replace the indigenous fermentation pathway(s) of E. coli. Jensen mentioned having specifically avoided carbon sources that provide types of growth other than anaerobic respiration (i.e. anaerobic fermentation). Thus a new strain would need to have the dehydrogenases required for anaerobic respiration, such as lactate, acetate, formate, or glycerol dehydrogenases. These dehydrogenases perform the first step to generating both proton motive force and provide an electron source in the quinol pool.

Competing sinks for electrons in E. coli where described as follows by Jensen [.]; “The bacterium always needs the process of oxidation involving the respiratory chain and usage of a quinone-pool to stay alive. To make this work, we need to somehow create a division between the respiratory chain which provides the electrons and the respiratory chain that provides the cell with energy.“ How can or should we integrate or separate electron transfer- and respiratory pathways? Could we separate these processes physically, or saturate the natural ‘stock’, or grow anaerobically? inhibitor ATP-synthase etc.; balance the quinone-pool or feed respiration (NADH, oa.)?

Several optional sources of carbon have been considered, pyruvate being one of the options. Questions posed with respect to pyruvate utilization, generating ethanol, acetyl-CoA (incl. ATP) and fumarate, are, amongst others, whether there are transporters present in order to relief the cell from reaction products; what determines the conversion of pyruvate to acetyl-CoA when sinks are changed for EET purposes, there not seemingly being theoretical reasons for respiring it to CO2.

Utilization of lactate as sole carbon source

E. coli, a facultative anaerobe, carries out mixed-acid fermentation of glucose in which the principal products are formate, acetate, D-lactate, succinate, and ethanol. During anaerobic growth, D-lactate is produced in order to recycle NADH produced by glycolysis. Cofactor NAD+ is generated, lactate secreted, the net supply per glucose being 2 ATP. This points to a first major issue in growth on lactate.

Enzymes involved in the conversion of lactate to pyruvate and vice versa are LldD, specific for D-lactate, soluble LdhA, an NAD-linked fermentative enzyme and Dld, a membrane-associated respiratory enzyme. Formation of D(-)lactate from pyruvate catalyzed by D-LDH is most likely unidirectional, which could mean pyruvate as a product in use of lactate could be (re)metabolized to lactate. Use of L-lactate and stimulation of E.coli to generate pyruvate in an enzymatic reaction using L-lactate specific L-LDH could pose an interesting solution to this potential problem.

The oxidation of DL-lactate by Escherichia coli has been shown to be catalyzed by at least two enzymes, one specific for L-lactate, the other specific for D-lactate. Both enzymes are present during aerobic and anaerobic growth on regular media. A higher level of both enzymes was produced in cells grown with lactate as growth medium, and the level of L-lactate dehydrogenase was markedly reduced by growth on glucose, pyruvate or succinate. The overall picture is an extremely complex one, where both enzymes are produced in equal amounts aerobically and anaerobically, enzyme levels are increased during growth on lactate, and the level of t-lactate dehydrogenase is markedly reduced by growth on glucose. Also, as Jensen reports, to date, increasing the number of conduits by transcriptionally upregulating mtrCAB has never increased iron oxide or electrode reduction, and it has not yet been determined why. As a possible explanation Jensen proposes that lactate oxidation by lactate dehydrogenase is rate limiting, inherently limiting the number of electrons delivered to the conduit.

In conversion of lactate to pyruvate, generation of ATP will be confined to a minimum and will thus affect growth. Aerobic growth on glucose followed by anaerobic generation of electron transport on lactate seems, summarizing, most feasible in development of a system in which extracellular electron transport is functional. Due to exposure to oxygen, enzymes functional in the tricarboxylic acid cycle (TCA cycle) will be expressed. (..) the assumption that fermentation will siphon electrons away from the electron conduit has not been proven; one could argue that a combination of fermentation and respiration could better support growth, and thus increase current out of the cell. Although I have done a series of experiments to determine what carbon source works best in our engineered E. coli strains, experiments using a mixture of these carbon sources with a fermentable sugar, such as glucose, may provide valuable insight into what impact fermentation has on anode reduction.

Redoxpotentials of elements central in electron transfer

Proteins of which the extracellular electron transport chain consists can be classified, in part, as cytochromes. Cytochromes are proteins containing one or several groups of heme, a porphyrrin structure able to bind iron.

The basics for electron transfer are in fact formed by the redoxpotentials of the relevant compounds, the consecutive intracellular shuttles shifting electrons from the original electron donor. the carbon source. eventually towards a terminal electron acceptor, being the counterelectrode. Troubling are the redoxpotentials of several consecutive cytochromes, starting with CymA and / or native NapC. If NADH is not reduced, it will build up in the cell, as proposed in the section Modeling, and will form a bottleneck in generation of EET.

CymA and NapC carry several heme groups, of which the spin states of bound iron determine the redoxpotentials, that can vary considerably and will thus determine to what extent reduction (i.e. transport) takes place. It must also be mentioned that the transfer of electrons in vivo is, to a certain extent, determined by the surroundings of the protein considered as well as the states of, amongst others, redox poules and might thus be quite different than potentials determined in vitro.

Challenges and follow-up in generation of extracellular electron transport