Team:Westminster UK/Model
Project
PET Breakdown - the long reaction chain through TPA
One long-term application goal we believe in is that the power of exo-electrogenic bacterial activity can one day be applied in the breakdown of plastic polymers. In this case, we will specifically focus on one of the most commonly used single use plastic polymers, PET.
In order to generate an accurate model of PET and TPA breakdown that would occur within a microbial consortium, the first important step is to identify the breakdown reactions taking place within the bacterial cells and determining the bottleneck reactions that are crucial for the speed of PET degradation.
In this modeling chapter, the reactions are laid out in figure 1
Notes:
- TphP is originally called TphC
- Oxaloacetate goes into the TCA cycle through citrate synthase
Ethylene glycol breakdown
Some of the work for Ethylene glycol breakdown was already Postulated pathway for the metabolism of ethylene glycol in Pseudomonas putida strains KT2440 and JM37. The enzymes and/or metabolites identified in response to ethylene glycol in KT2440 are depicted in black. Additional pathways identified in strain JM37 are shown in gray. Detailed descriptions of the pathways are given in the text (Bjorn et al, 2012).
By employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis, it is found that Pseudomonas putida KT2440 does not grow within 2 days of incubation, compared to Pseudomonas putida JM37 which can grow rapidly under the same conditions. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Postulated pathway for the metabolism of ethylene glycol in Pseudomonas putida strains KT2440 and JM37 is shown left (Bjorn et al, 2012).
PET to TPA
Source: Yoshida, Shosuke, et al. "A bacterium that degrades and assimilates poly (ethylene terephthalate)." Science 351.6278 (2016): 1196-1199
Native to: Ideonella sakaiensis 201-F6
Enzyme kinetics-
> When the supernatant of the cell culture was analyzed, MHET levels were extremely small this indicates a rapid turnover of MHET.
> PETase mRNA was by far the transcript of highest concentration compared to other enzymes. Sometimes reaching up to 41 times the concentration of other transcripts.
> PETase Kcat is around .8 sec and Km is 7.3 ± 0.6 μM
Conclusion:
PET to MHET is the limiting step; PETase is the worst performing enzyme.
TPA to PCA
Source: Sasoh, Mikio, et al. "Characterization of the terephthalate degradation genes of Comamonas sp. strain E6." Appl. Environ. Microbiol. 72.3 (2006): 1825-1832.
Native to: Comamonas sp. strain E6
Enzyme kinetics-
TBD
PCA metabolism
Source: Dagley, S., Geary, P.J. and Wood, J.M., 1968. The metabolism of protocatechuate by Pseudomonas testosteroni. Biochemical Journal, 109(4), pp.559-568.
Native to: Pseudomonas testosteroni
Enzyme kinetics-
No enzymes specifically mentioned, but a Km value of 0.046 mM was determined.
Kaiserslautern iGEM team HPLC analysis
The results presented by the Kaiserslautern iGEM team after their HPLC analysis of the composition of the media where their reactions were tested. Their results were as follows after 96 hours of incubation at 30C:
|
Sig |
Sample |
Sum of mAU*s |
mAU |
|
210 |
N4 PET 30 96 |
8300.52590 |
1166.49440 |
|
NADI PET 30 96 |
3.09962e4 |
2991.05604 |
|
|
UVM4 PET 30 96 |
9429.40875 |
1199.93671 |
|
|
255 |
N4 PET 30 96 |
422.77573 |
50.25511 |
|
NADI PET 30 96 |
328.29991 |
43.40195 |
|
|
UVM4 PET 30 96 |
432.70568 |
46.84119 |
|
|
300 |
N4 PET 30 96 |
180.67229 |
21.95549 |
|
NADI PET 30 96 |
115.38840 |
16.13500 |
|
|
UVM4 PET 30 96 |
168.42084 |
19.75307 |
Lowest and highest values for each Sig. are highlighted.
References:
Mazurkewich, S., Brott, A.S., Kimber, M.S. and Seah, S.Y., (2016). Structural and kinetic characterization of the 4-carboxy-2-hydroxymuconate hydratase from the gallate and protocatechuate 4, 5-cleavage pathways of Pseudomonas putida KT2440. Journal of Biological Chemistry, 291,(14), 7669-7686.
Mampel, J., Providenti, M.A. and Cook, A.M., (2005). Protocatechuate 4, 5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases.Archives of microbiology, 183, (2) 130-139.
Kamimura, N., Aoyama, T., Yoshida, R., Takahashi, K., Kasai, D., Abe, T., Mase, K., Katayama, Y., Fukuda, M. and Masai, E., (2010). Characterization of the protocatechuate 4, 5-cleavage pathway operon in Comamonas sp. strain E6 and discovery of a novel pathway gene. Appl. Environ. Microbiol.,76, (24), 8093-8101.
Sasoh, M., Masai, E., Ishibashi, S., Hara, H., Kamimura, N., Miyauchi, K. and Fukuda, M., (2006). Characterization of the terephthalate degradation genes of Comamonas sp. strain E6.Appl. Environ. Microbiol., 72, (3), .1825-1832.
Bjorn Mückschel,a Oliver Simon,b Janosch Klebensberger,a Nadja Graf,c Bettina Rosche,d Josef Altenbuchner,c Jens Pfannstiel,b Armin Huber,b and Bernhard Hauera (2012). Ethylene Glycol Metabolism by Pseudomonas putida. Applied and Environmental Microbiology, 78 (24), 8531–8539.
Kamimura, N. and Masai, E., (2014). The protocatechuate 4, 5-cleavage pathway: overview and new findings. In Biodegradative Bacteria (pp. 207-226). Springer, Tokyo. (https://zero.sci-hub.tw/2299/1d287427c5c2b447411f92f77ce5ed0e/kamimura2013.pdf)
MTR Pathway
Chemical products of metabolic pathways are always eventually transformed into electrical forms of energy through various electrochemical reactions. Such is the case for lactate, the main example of a product that is generated at the end point of PET breakdown in our modeled pathway.
A few main ways that exo-electrogenic bacteria harvest electric energy include the extraction of electrons from the cell, which can be concentrated at an electrode that creates a high electric potential difference against the intracellular environment of the cell. Another way electrons can be transferred is through oxidised mediator molecules that transfer them further to the electrode, such as NADH, FADH, quinones as well as a variety of other parameters and interactions that can ultimately influence electricity generation.
The goal of this aspect of the model is to address the main products that appear to influence the output most strongly out of all the reactions that are occuring.
Here we will build upon what is already known and somewhat researched by other teams, while including our newly introduced variables. The main focus of the theoretical analysis will include the following factors:
- NADH/H+ intermediates
- Reduction of oxidised mtr components via NADH
- Presence of oxygen / effects of transcription factors EtrA, RpoE and CRP
- Transfer of electrons from reduced mediator to the electrode
NADH intermediates
It is the biochemical processes inside the cell that are most responsible for a strong activity of the Mtr components. Depending on the type of the cell and its metabolic adaptivity to the environment, multiple pathways can contribute large amounts of electron donors. In this case we will focus on the glycolysis pathway, the TCA cycle and integrate PET breakdown enzymes that produce the NADH intermediates.
In this model, these include:
TPA breakdown:
TphB
Glycolysis:
Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH)
TCA cycle (includes one EG breakdown product):
Isocitrate dehydrogenase
α-ketoglutarate dehydrogenase
Malate dehydrogenase
This section will be focused on all the NADH (and NADPH, for TphB) intermediates, for which the general Michaelis-Menten equation can be used:
Where Vmax can also be represented as:
Where kcat represents the rate-limiting turnover number and E0 represents the enzyme concentration. The resulting equation, alternative to the first one would be, for each of the enzymes separately:
This applies to one breakdown sequence of a single MHET monomer breakdown.
Mtr oxidoreductive pathways
Consideration of electrochemical mediators is essential for the construction of an MFC, as the mediators pose another important bottleneck of the reaction process that leads to the transfer of electrons from bacteria onto the anode. Mediators generally tend to be small water-soluble molecules that are capable of being redox transformed and thus act as electron shuttles which enhance the kinetics of electron transfer. In MFC research, various molecules have been successfully tested as mediators, thus in order to have a valuable model some of the common mediators will be specified.
First important note to clarify is that there are two classes of mediators; endogenous and exogenous. Endogenous mediators are generated by the bacteria and can be secreted to the medium and then be oxidized at the electrode. Exogenous mediators, on the other hand, are redox molecules that are chemically synthesized and must be added into the anode chamber of the MFC in order to enable electron transfer from bacterial metabolic pathways to the anode.
- oneidensis contains endogenous mediators - namely, quinones, which work in between the inner and the outer membrane of the cell. Data for their efficiency was measured on a quinone analogue, anthraquinone-2,6-disulfonate (AQDS) by Han et al 2017. Their in situ data using the DT spectra to reflect the kinetics of ADQS reduction by c-Cytochromes in intact cells demonstrated that quinone efficiency greatly depends on the growth conditions the Shewanellas are in, with best efficiency measured at 30°C and a pH of 7.0. Calculations performed to obtain the yield resulting from the first step of lactate oxidation (which donates initial electrons to the MtrA-CymA complex) are described in the thermodynamic analysis section, as recorded from the Han et al research paper.
Thermodynamic analysis
Based on the AQDS reduction results by mutant and WT MR-1, the electron transfer pathway of AQDS reduction by MR-1 cells included intracellular electron transport (IET) and extracellular electron transport (EET) via inner membrane (In) and outer membrane (Ex) c-Cytochromes, that can be divided into the following three steps:
- IET from electron donor to In-Cytochromes
- IET from In-Cytochromes to Ex-Cytochromes or AQDS
- EET from Ex-Cytochromes to AQDS
In step 1, the electron donor (lactate) can be utilised by MR-1 and concomitantly IET then occurs from lactate to In-Cyts resulting in the redox transformation from In-Cyt (oxidised) to In-Cyt (reduced).
The reduction of In-Cyts is controlled by the utilisation efficiency of lactate, which is influenced by incubation conditions, including lactate concentration, pH, cell density and temperature.
In step 2, In-Cyt (reduced) can transfer electrons to Ex-Cyts, resulting in the formation of Ex-Cyt (reduced).
Because the AQDS may penetrate the cell membrane and contact the inner membrane and periplasmic electron transfer components (CymA and MtrA), In-Cyt (reduced) can also transfer electrons to AQDS (oxidised), resulting in the formation of AQDS (reduced).
In step 3, the electrons from Ex-Cyt (reduced) can be transferred to AQDS (oxidised) which is directly in contact with OM, resulting in the reduction of AQDS (oxidised) to AQDS (reduced).
The specific theoretical potential of lactate (E.C3H5O3-) can be calculated by the Nernst equation:
Where E0C3H5O3 represents the standard redox potentials of lactate, R is the ideal gas constant (8.3145 Jmol-1K-1), F is the Faraday constant (96,485 mol -1e-), and T is the temperature (298 K). From this equation, when the concentration of lactate increased, the specific theoretical redox potential of lactate decreased gradually, resulting in the increase of AQDS reduction capacity.
The electrons from the oxidation of lactate can be transferred through the following IET and EET processes which were mediated by the In-Cyts and Ex-Cyts. Since the key electron transfer component of In-Cyts and Ex-Cyts is a heme with an iron centre, the half reaction for In-Cyts and Ex-Cyts transformation is shown in the following reaction.
The specific redox potentials of hemes (EHeme) can be calculated by the following Nernst equation.
Where E0Heme represents the standard redox potentials of hemes. The driving force of the IET process (𝚫EIET) can be indicated as the following equation.
The electrons from Heme (reduced) in In-Cyts and Ex-Cyts can be transferred to AQDS resulting in the reduction into AHQDS- and AH2QDS as the following reactions.
The redox potentials of AQDS to AGQDS (EQ/QH) and AQDS to AH2QDS (EQ/HQ2) can be calculated from the following two equations.
Where E0Q/QH and E0Q/QH2 are the standard redox potentials of AQDS to AHQDS- and AQDS to AH2QDS, respectively. The reactions for 𝚫EIET and EQ/QH between In-Cyts/Ex-Cyts and AQDSOX may be influenced by speciation of AQDS as indicated by reactions for reduction of AQDS and AHQDS, which are strongly dependent on pH due to the pH-dependent redox potentials indicated by the two equations above.
While it is possible for AQDS to penetrate the cell membrane and react with In-Cytred, the mutant results suggested that the EET process played a dominant role in the AQDS reduction process. The driving force of the EET process (𝚫EEET) is indicated in the following equation.
References
iGEM Bielefeld (2013) https://2013.igem.org/Team:Bielefeld-Germany/Modeling#Mediator_Reduction
Han, R., Li, X., Wu, Y., Li, F. and Liu, T. (2017). In situ spectral kinetics of quinone reduction by c-type cytochromes in intact Shewanella oneidensis MR-1 cells. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 520, 505-513. Doi: https://doi.org/10.1016/j.colsurfa.2017.02.023
Coupling the reactions together - A three-way co-culturing consortium
The breakdown of plastic polymers and its coupling with foreign electron transduction pathways is a challenge that many research teams have attempted to take. Both within iGEM, such as Harvard (2016) that did preliminary research with composite BioBricks on PET plastic breakdown, and that made a potential application suggestion with their hardware design of the plastiback™ device (https://2016.igem.org/Team:Harvard_BioDesign/Achievements). Other teams, such as the IONIS iGEM team from 2019 which is attempting to do the same with a different plastic polymer, ethylene glycol, found in cigarettes, and has a plan to do this by co-culturing commonly used E. coli that would be modified to degrade ethylene glycol, together with S. oneidensis which would consume the intermediates and generate electricity (https://2019.igem.org/Team:Ionis_Paris).
In our project, we ended up focusing on PET, with the main motive of choosing this plastic being the ubiquitousness of iGEM teams focused on breaking down the PET polymer.
While most of our preliminary research was focused on looking for past teams that could offer us a good basis for PET breakdown or well characterised PET degradation enzyme Biobricks that we could use, we found that many current teams already work with the similar issue. Soon enough, we found a team from the Kaiserlautern University in Germany, which is in fact attempting to make a highly intriguing (and preferable) idea come true: Degrade PET using a type of single-celled green algae - Chlamydomonas reinhardtii, one of the most well researched green algae in biology.
We soon established contact with Kaiserslautern and agreed to work together in collaboration, with the main idea of the common goal being an integration of PET breakdown with the production of electricity, as detailed out in the two previous pages of the model.
In this section, we describe what we have thought through and done regarding the bacterial co-culturing aspect of PET breakdown, while pointing out the main obstacles we came upon and guidance on what can be done next.
The organisms
Together with Kaiserslautern, we are already one step ahead in determining which organisms would combine well together to form an integrated pathway that starts with PET breakdown and ends with electron transduction onto an anode surface.
The green microalgae, Chlamydomonas reinhardtii, would be the first-stage organism of this process, which has the ability to produce and secrete large amounts of PETase and MHETase enzymes into the medium which it is inhabiting. With PET plastic present in the medium, the enzymes would initiate the first bottleneck reaction of the pathway, producing MHET monomers from PET, and finally, terephthalic acid (TPA) and ethylene glycol (EG) as two monomeric products of MHET.
The next step of this co-culture comes down to the method of utilising these two breakdown products.
Ethylene glycol, as previously mentioned, is a good source of bioenergy that can be uptaken and utilised by specific microbes. Shewanellas are unfortunately not one of these, and neither are Chlamydomonas.
The other byproduct, TPA, is a large acidic byproduct with a benzene ring in the middle which makes it challenging to degrade, as can be seen in figure 1 of the TPA breakdown model page. It is therefore no wonder once again that not many other microorganisms are able to biodegrade it.
During our search for a solution, we have turned our focus towards Ideonella sakaiensis, the first bacterium identified for its ability to break down PET and solely survive by feeding on its monomeric compounds (Palm et al, 2019). This indicates that aside from being the first bacteria identified to produce PETase and MHETase, it also has the ability to uptake both TPA and EG. Setting aside the fact that it is not completely known how it does this, our model proposes an accurate presentation of the process, while the practical application of Ideonella being capable of the breakdown is still an ability that can be utilised in a co-culture.
|
Growth media |
Temperature |
pH |
Duration of duplication |
|
|
Chlamydomonas reinhardtii |
TAP HSM |
25 C |
6.7 |
12-75 h (depending on presence of light) |
|
Ideonella sakaiensis |
YSL / MSM with PET |
30-37 C |
7-7.5 |
20-40 days |
|
Shewanella oneidensis MR-1 |
LB agar, TSB, MSM |
25-30 C |
6.1-7 |
18 hours |
|
E. coli TOP-10 |
LB, SOB, SOC |
37 C |
6.1-7 |
4-6 hours |
|
E. coli BL21 |
LB, SOB, SOC |
37 C |
6.1-7 |
4-6 hours |
|
E. coli DH5-alpha |
LB, SOB, SOC |
37 C |
6.1-7 |
4-6 hours |
As it turns out, our university has already done substantial research on Ideonella, with more researchers studying its properties at exponential rates in the past year. Having easy access to the bacteria, we found that the Westminster MFC laboratory has already attempted assessing the possibility of co-culturing Ideonella together with Shewanella and the study being in a preliminary phase, awaiting for approval to do more research on it. Dr. Kyazze offered us some of their data and enabled us to find out that this co-culture would be possible.
Having this in mind, the current suggestion we have is a unified proposal for the three microorganisms to live together, and this is what we have attempted to build upon. Figure x provides a visualisation of how the organisms are expected to be cultured.
Figure 1: The three-way consortium: SO (Shewanella oneidensis), CR (Chlamydomonas reinhardtii) and IS (Ideonella sakaiensis), cultured in a ratio (10:20:1). Shewanella and Ideonella are cultured together on the anode (grey area), while Chlamydomonas is mobile in the liquid medium of the anode chamber.
When understanding how these grow together, we first identified that the Chlamydomonas, as both a mobile and highly metabolically active eukaryote, is capable of self-sustaining through photosynthesis while traveling around a medium. This makes it the perfect candidate for in-media culturing, where it would secrete PET degrading enzymes and further facilitate these crucial bottleneck reactions.
The use of Ideonella is rationalised by its ability to uptake both breakdown metabolites of PET degradation. It can utilise these for its own energy and proliferation, but apart from that it can potentially also help Shewanella grow faster, as evidenced by co-culturing papers of Shewanella and other bacteria (Rosenbaum et al, 2011).
Finally the Shewanella oneidensis, specifically the MR-1 strain that we are using, would be our exo-electrogenic bacteria of our choice, as we decided to work on it for our own research of exo-electrogenicity. Other exo-electrogenic bacteria could be more preferable for use, such as Shewanella putrefaciens, Geobacter sulfurreducens, Geobacter metallireducens, Aeromonas hydrophilia and Pseudomonas aeruginosa (Logan, 2009).
The provided ratio of culturing for the three cultures SO, CR and IS (as referenced from the figure) would be somewhere close to around 1:20:1. This rationalisation of 1:1 for Shewanella and Ideonella specifically roots from research findings from our University’s MFC laboratory, while the quantity of Chlamydomonas correlates for the quantity of plastic present in the medium.
In our co-culturing experiments, we worked with Shewanella and Chlamydomonas.
In the Dr. Kyazze’s experiment in MFCs, Shewanella and Ideonella were tested for activity with PET conditions.
For individual cultures we seed the MFCs at 10% v/v inoculum of OD 0.6. So for the mixture, the ratio would have been 5% v/v for each of the cultures.
|
Voltage/ Date |
Ideonella sakaiensis only |
Shewanella oneidensis |
Shewanella oneidensis and Ideonella sakaiensis |
|
|
20.07.2019 |
First date |
0 |
0 |
0 |
|
28.07.2019 |
After 8 days Before changing the media |
5.6 mV |
120 mV |
35 mV |
|
5.08.2019 |
After 16 days |
110.306 mV |
96.905 mV |
129.203 mV |
|
6.08.2019 |
After 17 days |
109.130 mV |
95.905 mV |
127.224 mV |
One final issue we will attempt to address concerns the growth of microalgae together with microbes. When consulting with Dr. Percy, we found that an occurence of the microalgae feeding on the microbes could happen, as can be seen in figure x.
References:
Palm, G. J., Reisky, L., Bottcher, D., Muller, H., Michels, E. A. P., Walczak, C., Berndt, L., Weiss, M. S., Bornscheuer, U. T. and Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10, 1717.
Rosenbaum, M. A., Bar, H. Y., Quasim, H. Y., Beg, Q. K., Segre, D., Booth, J., Cotta, M. A. and Angenent, L. T. (2011). Shewanella oneidensis in a lactate-fed pure-culture and a glucose-fed co-culture with Lactococcus lactis with an electrode as electron acceptor.
Wang, V. B., Yam, J. K., Chua, S. L., Zhang, Q., Cao, B., Chye, J. L., & Yang, L. (2014). Synergistic microbial consortium for bioenergy generation from complex natural energy sources. TheScientificWorldJournal, 2014, 139653.
Logan, B. (2009). Exoelectrogenic bacteri that power microbial fuel cells. Nature Reviews Microbiology, 7, 375-383.
Toyama, T., Kasuya, M., Hanaoka, T. et al. (2018) Growth promotion of three microalgae, Chlamydomonas reinhardtii, Chlorella vulgaris and Euglena gracilis, by in situ indigenous bacteria in wastewater effluent. Biotechnol Biofuels, 11, 176. https://doi.org/10.1186/s13068-018-1174-0
