Team:USTC/Design
Conduction system
1. Metal reductase (Mtr)
a. Overview
The goal of our conduction system is to transfer extracellular electrons into the cytoplasm and supplement engineered bacteria with extra electrons to enhance the productivity of bacteria. As the electron carrier during the extracellular electron transfer, Metal Reductase(Mtr) is the most important part in our conduction system. MtrCBA protein complex forms a tunnel across the outer membrane and builds up the expression path for electrons between outer membrane and cytoplasm. We heterologously expressed Mtr CAB in Escherichia coli(E.coli) and enabled E.coli to “eat” extracellular electrons and then synthesize more NADH for fermentation and production (shown in Figure 1).
Figure 1. Pathway of electron transfer in conduction system
(OM stands for outer membrane, IM indicates inner membrane, Q means quinone)
b. Components of Mtr System
Mtr respiratory pathway is composed of cytochromes and structural proteins found in Shewanella oneidensis strain MR-1, a facultative anaerobe. The complete Mtr pathway consists of OmcA, MtrA, MtrC, MtrB and CymA. Both MtrA and MtrC are decaheme cytochromes, with molecular weights of 38kDa and 75 kDa respectively. MtrB is a non-heme transmembrane β-barrel with a molecular weight of 76 kDa. OmcA is the outer membrane decaheme cytochrome and CymA is the membrane associated quinol oxidase (shown in Table 1).
Table 1[2]. Properties of MtrCBA and CymA
MtrA is localized to the periplasm and contacts with other periplasmic electron transfer proteins. MtrC is secreted to outer membrane and binds the membrane through an N-terminal lipid anchor. It is proposed that MtrB functions as a porin connecting periplasmic MtrA with outer membrane MtrC (shown in Figure 2). The electron transfer happens once both MtrA and MtrC insert into MtrB to allow direct heme-to-heme electron exchange.
Figure 2[2]. Localization of proteins of Mtr pathway
c. Electron Transfer Mechanism
In Shewanella oneidensis strain MR-1, the electron from intracellular reactions can be transferred out of the cell via the Mtr pathway. In details, electrons from the menaquinone pool are passed to CymA. MtrA, the periplasmic face of Mtr system, oxidizes the CymA and then transfers the intracellular electron to MtrC on the outer membrane. After obtaining the electron, reduced MtrC could react with oxidizing agent in the media such as solid metal, metal ion, mediators and transfer electrons to other substrates. (shown in Figure 3) The proteins involved in this pathway are cytochromes composed of polypeptide and heme which serves as electron carrier. The process of electron absorbance and emission is realized by the redox state change of Ferrous ion coordinated to porphyrin.
Figure 3. The path of electron transfer in Shewanella oneidensis
d.Reverse electron transfer and bio-cathode
In 2010, the feasibility of the E. coli transferring electron to the extracellular electrode via expression of only mtrC, mtrA, and mtrB was proved. In 2011, the functional reversibility of the Mtr pathway was proved in Shewanella oneidensis strain. Addition of fumarate to a film of S. oneidensis adhering to a graphite electrode poised at -0.36 V versus standard hydrogen electrode (SHE) immediately led to electron uptake. Electrons can be transferred reversely to the cytoplasmic CymA and then to FccA which catalyze the reduction of fumarate to succinate (shown in Figure 4). The find of reverse extracellular electron transfer suggests the possibility of bio-cathode which bacteria expressing Mtr pathway binds to and obtain electrons from. Once these electrons are transferred to the quinol pool located at the cytoplasm, these menahydroquinone(MQH2) may react with NADH-dehydrogenase. The extra source of electrons will promote the synthesis of NADH which is the typical substrate of [H]. In this way, cells turn to be more reductive with more NADH and pushes the processing of reductive biochemical reactions which often are key steps for synthetic reactions. These electrons provided by the cathode advance the potential productivity of engineered bacteria.
Figure 4. The path of reverse electron transfer in Shewanella oneidensis
e. Our Design
We chose Mtr pathway as the core of conduction system because it is the most commonly studied and best understood. And we supposed that E.coli could obtain electrons from the cathodes or mediators after expressing MtrCBA successfully. Subsequently, we conducted experiments that bolster the hypothesis and constructed our conduction system successfully.
Figure 5. The plasmid we designed in our conduction system
Figure 6. Conduction system and harvest system
f. Basic Principle of Three-electrode System
The working electrode(WE) is the electrode in an electrochemical system on which the reaction of interest is occurring. Common working electrodes can be made of inert materials such as Au, Ag, Pt, glassy carbon (GC) and Hg drop and film electrodes etc. For corrosion applications, the material of the working electrode is the material under investigation (which is actually corroding). The size and shape of the working electrode also varies and it depends on the application.
The counter electrode(CE) (also known as auxiliary electrode), is an electrode which is used to close the current circuit in the electrochemical cell. It is usually made of an inert material (e.g. Pt, Au, graphite, glassy carbon) and usually it does not participate in the electrochemical reaction. Because the current is flowing between the WE and the CE, the total surface area of the CE (source/sink of electrons) must be higher than the area of the WE so that it will not be a limiting factor in the kinetics of the electrochemical process under investigation.
The reference electrode(RE) is an electrode which has a stable and well-known electrode potential and it is used as a point of reference in the electrochemical cell for the potential control and measurement. The high stability of the reference electrode potential is usually reached by employing a redox system with constant (buffered or saturated) concentrations of each participants of the redox reaction. Moreover, the current flow through the reference electrode is kept close to zero (ideally, zero) which is achieved by using the CE to close the current circuit in the cell together with a very high input impedance on the electrometer (> 100 GOhm).
Figure 7. Scheme of a three-electrode system
The three-electrode cell setup is the most common electrochemical cell setup used in electrochemistry (see Figure 7). In this case, the current flows between the CE and the WE. The potential difference is controlled between the WE and the CE and measured between the RE (kept at close proximity of the WE) and S. Because the WE is connected with S and WE is kept at pseudo-ground (fixed, stable potential), by controlling the polarization of the CE, the potential difference between RE and WE is controlled all the time. The potential between the WE and CE usually is not measured. This is the voltage applied by the control amplifier and it is limited by the compliance voltage of the instrument. It is adjusted so that the potential difference between the WE and RE will be equal to the potential difference specified by the user. This configuration allows the potential across the electrochemical interface at the WE to be controlled with respect to the RE.
Reference:
[1] Pitts, K. E., Dobbin, P. S., Reyes-Ramirez, F., Thomson, A. J., Richardson, D. J., & Seward, H. E. (2003). Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA expression in Escherichia coli confers the ability to reduce soluble Fe (III) chelates. Journal of Biological Chemistry, 278(30), 27758-27765.[2] Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y., Cohen, B. E., Helms, B. A., ... & Ajo-Franklin, C. M. (2010). Engineering of a synthetic electron conduit in living cells. Proceedings of the National Academy of Sciences, 107(45), 19213-19218.
[3] Ross, D. E., Flynn, J. M., Baron, D. B., Gralnick, J. A., & Bond, D. R. (2011). Towards electrosynthesis in Shewanella: energetics of reversing the Mtr pathway for reductive metabolism. PloS one, 6(2), e16649.
[4] Shi, L., Squier, T. C., Zachara, J. M., & Fredrickson, J. K. (2007). Respiration of metal (hydr) oxides by Shewanella and Geobacter: a key role for multihaem c‐type cytochromes. Molecular microbiology, 65(1), 12-20.
[5] Ross, D. E., Ruebush, S. S., Brantley, S. L., Hartshorne, R. S., Clarke, T. A., Richardson, D. J., & Tien, M. (2007). Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. Applied and environmental microbiology, 73(18), 5797-5808.
[6] Edwards, M. J., Fredrickson, J. K., Zachara, J. M., Richardson, D. J., & Clarke, T. A. (2012). Analysis of structural MtrC models based on homology with the crystal structure of MtrF.
[7] White, G. F., Edwards, M. J., Gomez-Perez, L., Richardson, D. J., Butt, J. N., & Clarke, T. A. (2016). Chapter three-mechanisms of bacterial extracellular electron exchange. Advances in microbial physiology, 68, 87-138.
[8]EC08 A A N. Basic overview of the working principle of a potentiostat/galvanostat (PGSTAT)–Electrochemical cell setup[J]. Metrohm Autolab. BV, 2011: 1-3.
2. Cytochrome c maturation (Ccm)
a. Heme on MtrA&C
Our conduction system is composed of three proteins: MtrA, MtrB, MtrC. The two proteins MtrC&A are the member of cytochrome C family. Cytochrome C is a kind of vast protein family which involves in many redox biochemical reactions. Proteins belonging to this family generally contain at least a redox center as electron carrier. In terms of MtrC&A, the function of electron transfer relies on heme which is covalently bond to the polypeptide via thioether bonds (shown in figure 8). The component of heme includes a porphyrin and an iron redox center coordinated with the porphyrin. MtrC&A transfer electrons via the change of redox state of iron center (shown in table 2). Although the structure of MtrC and MtrA has not been solved, but the crystal structure for the MtrC homologue, MtrF has been solved. The structure of MtrF showed a novel jagged-cross heme structure (shown in Figure 9). Heme 10 is proposed to face the membrane and heme 5 is proposed to be the terminal reductase on solid surfaces. Heme 7 and heme 2 are proposed to reduce soluble iron or diffusible redox species. The main structure of MtrB is the β-barrel across the outer membrane so this protein is not the electron carrier during the transfer of electron but a chaperone to stabilize MtrC which is anchored to MtrB.
| Protein | Number of heme |
|---|---|
| MtrA | 10 |
| MtrB | n/a |
| MtrC | 10 |
Table 2[1]. Number of heme on Mtr CAB
Figure 8. Structure of heme c
Figure 9[1]. Detail of Mtr CAB's structure
b. Ccm A-H complex
Ccm is an operon on the chromosome of E.coli coding for the protein complex CcmA-H which is located on the inner membrane of the bacteria. MtrC and MtrA biogenesis requires the cytochrome c maturation (ccm) genes. The CcmA-H complex catalyzes the process of cytochrome c maturation which acts as a heme lyase for covalent attachment of protoheme IX to the apoprotein (shown in Figure 10). The conserved heme binding motif is CXXCH; the two cysteine thiols covalently bind the porphyrin, and the histidine acts as one of the axial ligands to the iron redox center. CcmE, a heme chaperone, covalently binds heme at a conserved His residue. CcmF, CcmH, and CcmG have all been implicated in the role of heme lyase, and together they catalyze the formation of thioether bonds between the apoprotein heme binding motif cysteines and heme b. Once these processes are complete, the axial ligands coordinate to the heme iron. In this way, heme is attached to the backbone of polypeptide and cytochrome c gets mature.
Figure 10. Function of Ccm
c. Our design:
Transcription of the genomic ccm operon in E.coli is repressed under aerobic conditions. To form mature MtrC&A under aerobic condition, we get the copy of Ccm gene from the genome of E.coli by PCR. Then we insert ccm operon to the recombinant pSB1C3 with constitutive promoter pTet (shown in Figure 11). We co-transform the recombinant pSB1C3 with ccm gene and pET28 with MtrCAB gene to establish mature MtrCAB system under aerobic condition. After the synthesis of Ccm A-H and MtrCAB, the Ccm and Mtr polypeptides are secreted into the periplasm. The Ccm system plays a role in translocating heme into the periplasm and then catalyzes the formation of thioether bonds that link hemes to two cysteine residues. Once these processes are complete, the axial ligands (typically histidine) are coordinated to the heme iron and the holocytochrome c is folded (whole pathway shown in Figure 12). MtrC&A get mature with the attachment of heme and Mtr protein complex acquire the ability of electron transfer.
Figure 11. Plasmid pTBC consists of pTet and gene ccm A-H
Figure 12[1]. Whole pathway of Ccm function and Mtr expression (the upper membrane is the outer membrane and the other is inner membrane)
References:
[1] Jensen, H. M. (2013). Engineering Escherichia coli for molecularly defined electron transfer to metal oxides and electrodes. University of California, Berkeley.[2] Jiang, X., Hu, J., Fitzgerald, L. A., Biffinger, J. C., Xie, P., Ringeisen, B. R., & Lieber, C. M. (2010). Probing electron transfer mechanisms in Shewanella oneidensis MR-1 using a nanoelectrode platform and single-cell imaging. Proceedings of the National Academy of Sciences, 107(39), 16806-16810.
[3] Sanders, C., Turkarslan, S., Lee, D. W., & Daldal, F. (2010). Cytochrome c biogenesis: the Ccm system. Trends in microbiology, 18(6), 266-274.
[4] Arslan, E., Schulz, H., Zufferey, R., Künzler, P., & Thöny-Meyer, L. (1998). Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of thecbb3Oxidase in Escherichia coli. Biochemical and biophysical research communications, 251(3), 744-747.
[5] Clarke, T. A., Edwards, M. J., Gates, A. J., Hall, A., White, G. F., Bradley, J., ... & Wang, Z. (2011). Structure of a bacterial cell surface decaheme electron conduit. Proceedings of the National Academy of Sciences, 108(23), 9384-9389.
Photocatalyst system
1.Overview
As we all know, the photosynthesis reaction which utilizes solar energy initiates the energy flow and the cycle of elements in the ecosystem. The essence of photosynthesis is that photosynthetic pigments are activated to a high energy state via the absorbance of light. This process initiates the transfer of electrons. In our artificial photosynthesis system, we combine E.coli with MtrCBA complex and CdS nanoparticles which function as a photo-catalyst. If electrons of CdS nanoparticles are activated to the conduction bond, these electrons are possibly transferred to the cytoplasm of E.coli via tunnels of MtrCBA complex (shown in Figure 13). In this way, electrons provided by CdS participate in cellular redox biochemical reactions and we obtain reduction products from these reactions.
Figure 13. Effect of CdS nanoparticles
2.Photo-catalyst--CdS nanoparticles
Semiconductors are a wide range of solid materials that have interesting properties. When external factors, like temperature, light and pressure change it responses to these changes by changing its electric resistance. As we know, semiconductors have special bond structures which have been fully studied by chemists. The bond structure of semi-conductors can be divided into two energy band, the valence bond and the conduction band, between which is the forbidden zone. The energy gap of the forbidden zone is usually 0.3-0.5eV.
Photo-semiconductors are a rather special kind of semiconductors. When absorbing the energy of light, the electrons can leap from valence band to forbidden bond, leaving a hole in valence band and a free electron in conduction band (shown in Figure 14). The electrons can move from interior to the surface of the solid, then be used to reduce chemicals in solvent. The hole can move to the surface and react with another outside electron sources to annihilate.
CdS is a well-studied semiconductor with an appropriate band structure and is suitable for photosynthesis. Many hybrid systems have been developed by scientists. Early this year, Peidong Yang and his colleagues have developed a new hybrid system containing the non-photosynthetic CO2-reducing bacterium Moorella thermoacetica and its biologically precipitated CdS nanoparticles. In this system, the precipitation of CdS by M. Thermoacetica was initiated by the addition of Cd(NO3)2 to an early exponential growth culture of glucose-grown cells supplemented with cysteine.
Inspired by his work, we decided to use CdS nanoparticles in our work. Equipped with metal electrode, CdS may well transfer the electrons from electrode to the surface of the bacteria while giving light.
Figure 14. Electron excitation in CdS nanoparticles
3.Synthesis of CdS nanoparticles—CysDes
In our photosynthesis system, CdS nanoparticles are the vital component which undertake the task of emitting electrons. Because of the specific scale of CdS particles, we choose to synthesis these particles in a biological way. We enable E.coli to reduce the thiolate group of cysteine to S2-. Once S2- are secreted to the media containing Cd2+, insoluble CdS precipitating on the surface of E.coli in a nanoscale and CdS nanoparticles get attached to the bacteria (shown in Figure 15).
The enzyme that we use to reduce cysteine is cysteine desulfhydrase (CysDes) from Treponema denticola. CysDes is capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This 46kDa cysteine desulfhydrase results in the following Michaelis-Menten kinetics: Km =3.6 mM and kcat=12 s-1. Cystathionine and S-aminoethyl-L-cysteine are also substrates for the protein.
In our project, we heterologously express CysDes in E.coli. According to some reports[2], when grown in defined salts medium supplemented with cadmium and cysteine, E.coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. This fact suggests the possibility of generating photoactive nanoparticles by microbial formation of cadmium sulfide. We assume that these CdS nanoparticles could precipitate and attach to the surface of our bacteria, so they could serve as an electron source with the light excitation.
Figure 15. Scheme of the synthesis process of CdS nanoparticles
4.Construction of our photocatalyst system
The sequence of CysDes is downloaded from genebank and we synthesize this gene by Integrated DNA Technologies, Inc. (IDT). We choose pSB1C3 with promoter pLuxR as the expression vector and insert cysdes gene to it. Then we transform the plasmid containing cysdes to BL21 and induce the expression by adding AHL(N-Acyl homoserine lactones) (shown in Figure 16). To confirm the expression, we analysis the enzyme activity in vitro by measuring the amount of S2-. To ensure the formation and attachment of CdS nanoparticles, we add appropriate amount of Cd2+ and observe the form of CdS particles and the surface of E.coli via the transmission electron microscopy.
Figure 16. Plasmid pLCys consists of pLuxR and gene CysDes
References:
[1] Chu, L., Ebersole, J. L., Kurzban, G. P., & Holt, S. C. (1997). Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities. Infection and immunity, 65(8), 3231-3238.[2] Wang, C., Lum, A., Ozuna, S., Clark, D., & Keasling, J. (2001). Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene. Applied microbiology and biotechnology, 56(3-4), 425-430.
[3] Sakimoto, K. K., Wong, A. B., & Yang, P. (2016). Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production. Science, 351(6268), 74-77.
Harvest system
1. Overview
On the basis of Conduction system and Photocatalyst system, our engineered E.coli is now able to utilize photo-excited electrons to improve its own reducing power. In our final module, the harvest system, we attempt to combine Photo-Electro E.coli with a biosynthetic pathway where NADH is required. We hope the gained reducing power can help improve the efficiency of synthetic reactions.
Figure 17. The three systems in our project
2. NADH-depend alcohol dehydrogenase KmAdh
For our project, we introduce an NADH-depend enzyme called KmAdh, which is an alcohol dehydrogenase (ADH) from Kluyveromyces marxian. Experiments have shown that KmAdh heterologously expressed in E.coli can efficiently catalyze the conversion of acetaldehyde to ethanol in the existence of NADH.
Figure 18. The function of KmAdh
Ethanol, as a very important material in both food and chemical industry, is of enormous demand. During a long period, yeast fermentation is the major way to produce ethanol, but in recent years people are trying to engineer ethanol pathway into E.coli, for E.coli is a commonly used chassis for biosynthesis. We choose an ADH to validate the function of our system because it is an essential enzyme for genetic engineering of ethanol production in E.coli, and we hope our project can help this enzyme perform better.
To be more detailed, we construct a plasmid containing the segment which encodes KmAdh, using pET22b with T7+lacO promoter as the expressing vector. We transform this plasmid into BL21 and inducing it with IPTG, equiping our PELICAN with the ability to convert acetaldehyde to ethanol. Now with the help of Conduction system and Photosynthesis system, NADH can be recycled quickly and then be fully utilized in the reduction reaction, thus the efficiency of this conversion will be largely improved.
Figure 19. The plasmid we designed in our harvest system
3. Universal biosynthesis platform
E.coli is a commonly used engineering bacteria, in which biosynthetic pathways of many important chemical material are constructed into biobricks. In fact, our PELICAN is able to serve as a standardized platform which can load all those biobricks and help promote their function.
To prove its universal property, we also introduce another enzyme called CmCR, which is a carbonyl reductase that can reduce ethyl 4-chloro-3-oxobutyrate (COBE) to Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important and valuable chiral intermediate. Traditional chemical way to synthesize (S)-CHBE always leads to a high cost and low enantiomeric excess value. Biotransformation seems to be a good way for its convenience and high specificity, but how to sustain a continuous supply of the cofactor, NADH, is still an essential problem. We hope our PELICAN can be an effective synthetic platform which can realize the regeneration of NADH, and finally achieve the goal to both lower the cost and improve the efficiency.
Figure 20. The function of CmCR
Reference:
[1]Liang, J. J., Zhang, M. L., Ding, M., Mai, Z. M., Wu, S. X., Du, Y., & Feng, J. X. (2014). Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization. BMC biotechnology, 14(1), 45.[2]Deng, M. D., Severson, D. K., Grund, A. D., Wassink, S. L., Burlingame, R. P., Berry, A., ... & Rosson, R. A. (2005). Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metabolic engineering, 7(3), 201-214.
[3]He, Y.C., Tao, Z.C., Zhang, X., Yang, Z.X., Xu, J.H., 2014a. Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH-dependent reductase from E. coli CCZU-K14. Bioresour. Technol. 161, 461–464.
