Poster: TU_Kaiserslautern

Microdestruction Against Micropollutants in Wastewater

Sarah Abdul-Mawla*^,**, Richelle Avers*, Emily Becker*^,**, Daniel Brady*, Nicolas Freche*^,**, Stefanie Heinrich*, Allyssa Hinkle*^,**, Linda Müller*^,**, Helena Schäfer*^,**, Yannik Schermer*^,**
Nicole Frankenberg-Dinkel, Michael Schroda, Felix Willmunnd
*TU Kaiserslautern iGEM 2020, Technische Universität Kaiserslautern
**Everyone mentioned above contributed equally to the poster. The names of the team members were ordered alphabetically.

Abstract

Micropollutants are a massive concern in wastewater treatment, as their accumulation can seriously impact ecosystems. Anti-inflammatory medications such as Ibuprofen or Diclofenac are primary examples of micropollutants becoming an ever-growing problem through patient overuse and relaxed disposal practices. The enzyme laccase has been shown to chemically deactivate Diclofenac, leading to functional detoxification. Different laccase genes (MarLac, from uncultivated marine bacteria, and BaLac, from a mutant Botrytis aclada) were cloned and transformed into both our control bacterium, Escherichia coli, and primary biotechnological organism, the green algae Chlamydomonas reinhardtii. Produced laccases would be incorporated into a bioreactor set up. Our project will use synthetic biology in an innovative and cost-effective way to produce a self-sufficient system. We want to reduce the need for specialized and still experimental equipment with easy integration into existing sewage treatment plant systems in order to create cost-effective and efficient approaches to a cleaner and healthier environment.

Introduction

Currently, many pharmaceuticals and other micropollutants enter the environment through improper disposal and overuse. Many of these substances are understudied and may pose a risk to marine organisms, microorganisms, and their ecosystems.¹ However, only a small fraction of these micropollutants are subject to regulation by government entities.¹ Prolific examples of these substances are anti-inflammatory drugs like Diclofenac and Ibuprofen.

Laccases are multi-copper oxidoreductases which oxidize their substrates either directly or indirectly. In direct oxidation, the substrate interacts with the laccase‘s copper cluster. The indirect oxidation involves a mediator, which is oxidized by the laccase. The mediator then oxidizes the substrate.² An overview of the direct reaction is shown in Fig. 1.

Fig. 1: Overview for the direct reaction mediated by a laccase. One electron is transferred from the substrate to the copper cluster, turning the substrate into a radical cation.² Some toxic substances and pharmaceuticals are shown as example substrates.^3,4 After four electrons have been transferred to the enzymes copper cluster, it binds molecular oxygen. Two consecutive two-electron reductions then form two water molecules, putting the enzyme back into its oxidized form.^2,5

References

(1) Rogowska, J.; Cieszynska-Semenowicz, M.; Ratajczyk, W.; Wolska, L. Micropollutants in Treated Wastewater. Ambio 2020, 49 (2), 487–503. https://doi.org/10.1007/s13280-019-01219-5.

(2) Agrawal, K.; Chaturvedi, V.; Verma, P. Fungal Laccase Discovered but yet Undiscovered. Bioresour. Bioprocess. 2018, 5 (1), 4. https://doi.org/10.1186/s40643-018-0190-z.

(3) Hahn, V.; Meister, M.; Hussy, S.; Cordes, A.; Enderle, G.; Saningong, A.; Schauer, F. Enhanced Laccase-Mediated Transformation of Diclofenac and Flufenamic Acid in the Presence of Bisphenol A and Testing of an Enzymatic Membrane Reactor. AMB Expr 2018, 8 (1), 28. https://doi.org/10.1186/s13568-018-0546-y.

(4) Kittl, R.; Mueangtoom, K.; Gonaus, C.; Khazaneh, S. T.; Sygmund, C.; Haltrich, D.; Ludwig, R. A Chloride Tolerant Laccase from the Plant Pathogen Ascomycete Botrytis Aclada Expressed at High Levels in Pichia Pastoris. Journal of Biotechnology 2012, 157 (2), 304–314. https://doi.org/10.1016/j.jbiotec.2011.11.021.

(5) Zerva, A.; Simić, S.; Topakas, E.; Nikodinovic-Runic, J. Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019). Catalysts 2019, 9 (12), 1023. https://doi.org/10.3390/catal9121023.

Our Goals

Fig. 1: Overview of our project goal. Dark Red: Diclofenac, an example micropollutant. Pink: Deactivated Diclofenac. Yellow: Our produced laccase. Our bioreactor allows our genetically modified Chlamydomonas reinhardtii to stay safely secure during the treatment process.

Inspiration

In Germany, the painkiller Diclofenac accumulates in fresh water as a micropollutant.
Other micropollutants such as antibiotics are more problematic throughout the rest of the world.
Current water treatment systems are not suitable for detoxifying all micropollutants. ¹

Fig 1: Proportions of medicinal products detected in the water and their mean concentrations (a) Global, (b) Europe, (c) North America, (d) Asia. Each point outward on the radial axis (y) stands for 10^y of the median concentration in ng L^–1.²

The unicellular green alga Chlamydomonas reinhardtii appears a well-suited chassis for the secretion of laccases, since it grows in fresh water, is non-toxic, and produces oxygen via photosynthesis that can serve as electron acceptor for oxidation reactions catalysed by laccases.
Laccases oxidize a wide range of phenolic micropollutants or amines.³
We chose two laccases, one from Botrytis aclada (BaLac) with a high redox potential and residual activity at pH 6.5,⁴ the other from a marine bacterium (marLac) with lower redox potential and high activity at pH 7.⁵
Both promise a high potential for bioremediation in a wastewater treatment plants due to their thermostability and stability in neutral pH wastewaters.

Fig. 2: Activity of the BaLac WT (line) and BaLac L499F mutant (circle) at different pH-levels. The used substrate was ABTS.⁴

References

(1) Deutscher Bundestag. Chemischer Zustand der Gewässer in Deutschland. August 26, 2019.

(2) Fig. 1 based on: Hughes, S. R.; Kay, P.; Brown, L. E. Global Synthesis and Critical Evaluation of Pharmaceutical Data Sets Collected from River Systems. Environ. Sci. Technol. 2013, 47 (2), 661–677. https://doi.org/10.1021/es3030148. edited with BioRender

(3) Kittl, R.; Mueangtoom, K.; Gonaus, C.; Khazaneh, S. T.; Sygmund, C.; Haltrich, D.; Ludwig, R. A Chloride Tolerant Laccase from the Plant Pathogen Ascomycete Botrytis Aclada Expressed at High Levels in Pichia Pastoris. Journal of Biotechnology 2012, 157 (2), 304–314. https://doi.org/10.1016/j.jbiotec.2011.11.021.

(4) Scheiblbrandner, S.; Breslmayr, E.; Csarman, F.; Paukner, R.; Führer, J.; Herzog, P. L.; Shleev, S. V.; Osipov, E. M.; Tikhonova, T. V.; Popov, V. O.; Haltrich, D.; Ludwig, R.; Kittl, R. Evolving Stability and PH-Dependent Activity of the High Redox Potential Botrytis Aclada Laccase for Enzymatic Fuel Cells. Sci Rep 2017, 7 (1), 13688. https://doi.org/10.1038/s41598-017-13734-0.

(5) Yang, Q.; Zhang, M.; Zhang, M.; Wang, C.; Liu, Y.; Fan, X.; Li, H. Characterization of a Novel, Cold-Adapted, and Thermostable Laccase-Like Enzyme With High Tolerance for Organic Solvents and Salt and Potent Dye Decolorization Ability, Derived From a Marine Metagenomic Library. Front. Microbiol. 2018, 9, 2998.

Simulation

The pH of the solution has a huge effect on the activity of the reacting enzyme.¹ The pK_a value of laccase and the charge of the side chains were analysed at pH 5 and 7. For this investigation the software DelPhiPKa was used.^2,3

Fig. 1: Charge of the histidine residues in the neighborhood of the copper ions as a function of pH.

Tab. 1: pK_a and charge calculation of the histidine residue at pH 5 and 7.

	Residue	pKa	Charge at pH 5	Charge at pH 7
T1 Copper	His 463	6.82	0.9959	0.5098
	His 531	6.94	0.9964	0.6100
T3 Copper	His 126	7.22	0.9666	0.6678
	His 168	5.05	0.5338	0.0932
	His 527	5.19	0.5488	0.1369
T3 Copper Adjacent	His 124	5.32	0.5598	0.1100
	His 170	4.89	0.5135	0.1654
	His 466	6.80	0.8774	0.4865
	His 525	4.85	0.4898	0.1778

The results for pH 5 and 7 are compiled in Tab. 1. The section labeled T3 Copper Adjacent lists the four histidine residues near the T3 Copper which could possibly impact the resulting charge. These results demonstrate there is a higher overall charge at pH 5. It can be assume that the histidine on the T3 coppers pulls the electron from the cysteine much more quickly, so the reactivity is higher.

References

(1) Alexey V Onufriev and Emil Alexov: Protonation and pK changes in proteinligand Binding. Quarterly Reviems of Biophysics, 2013, 46 (2), 181–209.

(2) Wang, L.; Li, L.; Alexov, E. P K a Predictions for Proteins, RNAs, and DNAs with the Gaussian Dielectric Function Using DelPhi p K a: Predicting p K a’s of Biomolecules. Proteins 2015, 83 (12), 2186–2197. https://doi.org/10.1002/prot.24935.

(3) Wang, L.; Zhang, M.; Alexov, E. DelPhiPKa Web Server: Predicting PKa of Proteins, RNAs and DNAs. Bioinformatics 2016, 32 (4), 614–615. https://doi.org/10.1093/bioinformatics/btv607.

Methodology

C. reinhardtii and E. coli (green and red box respectively): Construction of in vitro synthesized, codon-optimized versions of BaLac and marLac via Modular Cloning for C. reinhardtii and conventional cloning for E. coli.

Both organisms (purple box): Transformation into desired chassis, cultivation in liquid medium, purification and detection of produced proteins via SDS-PAGE and immunoblotting.

Activity Assay (blue box) via ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay and HPLC (High Performance Liquid Chromatography).

Fig. 1: Overview of our project methodology.

Results

C. reinhardtii Results

Fig. 1: Proof of cytosolic expression of BaLac (left) and marLac (right) in C. reinhardtii via immunoblotting. Red boxes indicate regions with the expected molecular masses.

E. coli Results

Fig. 2: Purification of BaLac with glutathione affinity chromatography. Red boxes indicate BaLac on a Coomassie-stained SDS gel and on an immunoblot decorated with an antiserum against GST. The expected mass of the protein is 61.6 kDa.The marker shows the molecular weight in kDa.

Assay Results

Proteins produced in E. coli were tested in ABTS assays.

Fig. 3: E. coli strain BL21 (DE3) ABTS assay performed at pH 4 in phosphate-citrate buffer. The positive control is Trametes versicolor laccase purchased from Sigma.

Fig. 4: HPLC analysis of Diclofenac oxidation by laccases. The red boxes indicate the elution peak of Diclofenac at different timepoints after the addition of laccase from T. versicolor (left) and from BaLac produced in E. coli (right).

Conclusion

BaLac produced in E. coli BL21 (DE3) was active and degraded Diclofenac according to our ABTS activity and HPLC results
Expression of marLac in E. coli resulted in low protein yields (not shown)
BaLac is more stable at a neutral pH than other available laccases¹
Similar to the results with E. coli, BaLac could be expressed effectively in C. reinhardtii, while marLac was hardly detectable; neither laccase could be secreted (not shown)

References

(1) Scheiblbrandner, S.; Breslmayr, E.; Csarman, F.; Paukner, R.; Führer, J.; Herzog, P. L.; Shleev, S. V.; Osipov, E. M.; Tikhonova, T. V.; Popov, V. O.; Haltrich, D.; Ludwig, R.; Kittl, R. Evolving Stability and PH-Dependent Activity of the High Redox Potential Botrytis Aclada Laccase for Enzymatic Fuel Cells. Sci. Rep. 2017, 7 (1), 1–13. https://doi.org/10.1038/s41598-017-13734-0.

Outlook

Implementation of the bioreactor

Flexible options for placement of the bioreactor within current treatment plant models e.g. sand trap

Containment must be transparent and have a gas supply → in the Kaiserslautern wastewater treatment plant there is a gas generator beneath the sand trap

Implementation Regulations

Regional State Authority interview
Finalized a testing scheme for a filtration system
Designed pretests to verify our system in all seasons
Understanding differing regulations between regions is key

Future iGEM teams

Detailed methods and expert advice
can be found on our Partnership Page

Fig. 1: Implementation in a wastewater treatment plant. Algae will be cultivated in a bioreactor next to a wastewater basin. Laccase enters the sand trap through the filtration system.

Fig. 2: Filtration system. Bioreactor contents are filtered in counter current principle. Verification of effective containment of our modified algae by plating dialyzed water on an agar plate to confirm that there is no growth.

Partnership with Darmstadt and Stuttgart

Teamed up with other laccase-based projects
Compared our projects
Collected literature and experts for future laccase-based projects

→ Coming together as a community

⇓

Working together to create something greater than the sum of its parts!

Fig. 1: The Oxiteers. They united to render problematic chemical compounds less toxic for us via oxidation.

Integrated Human Practice

Discussed logistics of our experiments with our advisors
Collaborated with other iGEM teams
Reached out to experts in science, law and engineering

→ We gained experience and confidence

⇓

Working together to create something greater than the sum of its parts!

Fig. 1: Our Wastewater Group meetings. With Team GenerationMendelBrno, Team Stuttgart, Team Aalto-Helsinki, Team UZurich, and Team TU Darmstadt.

Education

Organized a workshop for American students
Facilitated a field trip to a local water department & wastewater treatment plant
Students had to design a filtration experiment

→ Encouraging scientific literacy in children

⇓

Working together to create something greater than the sum of its parts!

Fig. 1: The Filtration Experiment with US students. Students tested their water samples and regularly recorded observations as the experiment progressed.

Team:TU Kaiserslautern/Poster

Poster: TU_Kaiserslautern