Team:TU Delft-Leiden/Project/Life science/EET/theory

Context

In the wet lab, we integrated the Electron Transport pathway of Shewanella oneidensis into Escherichia coli . Here you can find information with respect to literature consulted regarding implementation of the Electron Transport pathway in E. coli.

To facilitate extracellular electron transport in E. coli we genetically introduced a heterologous electron transport pathway of the metal-reducing bacterium S. oneidensis. The electron transfer pathway of S. oneidensis is comprised of C-type cytochromes that shuttle electrons from the inside to the outside of the cell [1]. As a result, this bacterium couples the oxidation of organic matter to the reduction of insoluble metals during anaerobic respiration. There are several proteins that define the route for the electrons and thus are the major components of the electron transfer pathway (see figure 1). Our key-player proteins are CymA, an inner membrane cytochrome, MtrA, which is a periplasmic decaheme cytochrome, MtrC, an outer membrane decaheme cytochrome and MtrB, an outer membrane β-barrel protein.

Now we have our so called Mtr electron conduit, but it will not function unless the multiple post-translational modifications are correctly carried out. The cytochrome C maturation (Ccm) proteins help the conduit proteins to mature properly by providing them with heme, which is one of the requirements to carry and transfer electrons [2]. The step-by-step assembly of the Mtr protein complex is described in more detail in Deterministic Model of EET Complex Assembly and Integration of Departments, subsection ‘Relevant details of the extracellular electron transport system.

Jensen et al.(2010) have described a genetic strategy by which E. coli was capable to move intracellular electrons, resulting from metabolic oxidation reactions, to an inorganic extracellular acceptor by reconstituting a portion of the extracellular electron transfer chain of S. oneidensis [3]. However, bacteria expressing the Mtr electron conduit showed impaired cell growth. To improve extracellular electron transfer in E. coli, Goldbeck et al. used an E. coli host with a more tunable expression system by using a panel of constitutive promoters. Thereby they generated a library of strains that separately transcribe the Mtr- and Ccm operons. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu (colony forming unit), rather than the strain with more MtrC and MtrA present [2]. In the Module Electron Transport we aimed to reproduce the results reported by Goldbeck et al. in a BioBrick compatible way. To our knowledge we are the first iGEM team that successfully BioBricked the Mtr pathway. On top of that, we have succeeded to BioBrick mtrCAB under control of a weakened T7 promoter with the lac operator (T7 lacO) and the Ccm cluster under control of the pFAB640 promoter, a combination that was found to generate the largest maximal current [2].

A Biosensor based on the Shewanella oneidensis Electron Transport pathway

Microbial Fuel Cell (MFC) based systems like the S. oneidensis Electron Transfer pathway are already introduced to the field of biosensors [4]. An arabinose inducible promoter system was used as proof of principle in the above mentioned case. These results clearly showed that the current production depends on the addition of arabinose in a linear fashion. Therefore we believe that the implementation of the Electron Transfer pathway in E. coli has potential to develop itself as a quantitative and inexpensive biosensor.

Improved parts lead to Extracellular Electron Transport

E. coli strains expressing the extracellular electron transfer complex display limited control of MtrCAB expression. In addition, these strains show impaired cell growth [2]. Use of a weakened T7 lacO promoter upstream of the mtrCAB cluster was shown to optimize the MtrCAB expression and reduce morphological perturbations [2]. Therefore we aimed to improve the MtrCAB BioBrick BBa_K1172401 of the Bielefeld 2013 team by adding the weakened T7 lacO promoter. While characterizing their BioBrick, we could not detect the coding sequence of MtrCAB by restriction analysis nor sequencing. Therefore we started from scratch to clone the MtrCAB genes under control of the weakened T7 lacO promoter. We confirmed the sequence, and were able to show extracellular electron transport using our own mtrCAB BioBrick BBa_K1316012 (see figure 2).

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 .

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 an electronegative acceptor. This reduced acceptor donates the electrons to an following acceptor which is even more electronegative, 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. The released energy from the transfer of electrons from donor to acceptor is used to generate a proton gradient across the mitochondrial membrane by the pumping of protons into the intermembrane space, which produces 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.

Competing sinks for electrons in E. coli where described as follows by Jensen [3]; “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? First thoughts on this matter included the possible physical separation of the processes oxidative phosphorylation and extracellular electron transport, the saturation of the natural ‘stock’, or addition of an inhibitor of ATP-synthase. 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 oxygen 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. The formation of potentially toxic intermediates will have to be taken into account. Also, 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.

Several optional routes for generation 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. 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 carbon dioxide? 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 in order to prevent generation of ATP via this route. Glycerol, for example, is a potential source of carbon. However, there is a possibility of 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), although she also points to the fact that "(..) 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."

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. 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.

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: Flux Balance Analysis of the EET Module 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

Major concept of interest is to what extent generation of ATP for growth might take place, however, it must be considered if that is the actual objective – an in-field plug-and-play biosensor used in, for example, the laboratory, might be considered a one-use-only device; in that case, it is of less importance to have proliferating cells. It might also be of interest to consider the possibility that once the EET has been saturated, the culture can be shifted towards aerobe glucose metabolism in order to get rid of the overflow of NADH. As the setup for measurements of current has been successfully implemented by this team, experiments might be executed in order to determine carbon sources maximizing EET. Also, in order to get a grip on use of D- and / or L-lactate, media could be measured after growth for the ratio of these isomers. FBA is per definition steady state; closed experimental system; no growth or flux. 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?

Determination of bottlenecks must be continued via carefully designed experiments. Are there, for instance, alternatives to NapC and / or can we increase NapC transcription? What is the impact of use of the native versus an engineered cytochrome C maturation system? Is there an influence of heme availability on the system? Is it possible and useful to increase the amount of type-II secretion systems present? Can we optimize transport and conductivity by including electron shuttles or mediators, ie. riboflavins? (possible) native transmembrane electron transport E.coli

An interesting final thought centers on redox potentials. As mentioned in the section ‘Redoxpotentials of elements central in electron transfer’, these potentials, resulting from differing spin states of iron, might pose a problem in transfer of electrons. Moving to a whole different section of Chemical Biology, redox potentials might in fact be adjusted. Reactivity of the iron center in heme depends on the coordination of iron by its ligands. Ligand chemistry, changing in the first coordination sphere, could decrease the overall potential of (for instance) CymA. At this point in time, this type of chemistry is not relevant for iGEM 2014. In a near future, however, it may very well be.

References

1.Yang et al., Bacterial extracellular electron transfer in bioelectrochemical systems. Process Biochemistry. 47, 707–1714 (2012)
2. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli ACS Synth. Biol. 2 (3), pp 150–159 (2013)
3. H.M. Jenssen et al., Engineering of a synthetic electron conduit in living cells. PNAS vol. 107 no. 45 (2010)
4. F. Golitsch et al., Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes. Biosensors & bioelectronics. Vol.47, pp.285-91 (2012) 5. D. Chang et al., 1999. Homofermentative production of D- or L-Lactate in Metabolically Engineered Escherichia coli RR1. Applied and Environmental Microbiology 65(4):1384-1389 6. G. Unden and J. Bongaerts, 1997. Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. Biochimica et Biophysica Acta 1320:217-234