Overview
Our iGEM team sets out to engineer and express various laccase constructs originating from the organisms Coriolopsis trogii, Trametes hirsuta, Trametes pubescens, and Trametes versicolor. The goal was secretory and extracellular production of these enzymes using a signal peptide in the yeast Pichia pastoris, purify these laccases, assess, and compare their enzymatic activities and evaluate their potential for degrading NSAIDs (non-steroidal anti-inflammatory drugs). To achieve our goal we utilized several methods, including Golden Gate Assembly, heat shock transformation, electroporation, and different fermentation strategies.
Cycle A - The cloning cycle
Design
We selected six laccases from the organisms Coriolopsis trogii, Trametes hirsuta, Trametes pubescens, and Trametes versicolor after research on their potential for degrading NSAIDs. Alongside we did all cloning steps using GFP as a control. These sequences were codon optimized for expression in Pichia pastoris and synthesized by Integrated DNA Technologies (IDT). We included a pre-Ost1-pro-α secretion factor from Saccharomyces cerevisiae, a TEV site, and a His-tag, along with fusion sites for Golden Gate Assembly. It was decided that the His-tag and TEV site should be added to the C-terminal end of our enzymes. A protein structure prediction using AlphaFold showed that the N-terminus would be internalized, increasing the risk of misfolding. To further mitigate that risk on the C-terminus, we designed primers to remove TEV site and His-tag.
Build
After receiving the DNA, we performed PCR to remove the cleavable His-tag, to have two versions of each laccase, with and without tags. We cloned them into our BB1_FS_23 vector, followed by heat shock transformation of Escherichia coli for amplification. The subsequent selection on Kanamycin plates was successful, and plasmid DNA extraction delivered a good yield. We then performed Golden Gate Assembly with BB2_L_AB vector assembling the genes with a pGAP promoter and RPS25Att terminator, forming a functional expression cassette. We then performed Golden Gate to assemble a Pichia pastoris compatible expression vector using the 100_BB3rN_Fs1_Fs4 plasmid, which was used in heat shock transformation of E. coli. After amplification using the bacteria, plasmid DNA extract and electroporation transformation of Pichia pastoris, we finally had cells that, in theory, produce our laccases.
Test
Colony PCR was performed after every transformation to check for successful take up of the plasmid. Furthermore, extracted plasmid DNA was sent in for sequencing to double check for mutations. When examining the sequencing results, we noticed that for two of our laccases, from the organism Trametes pubescens, the sequence length was off. It turns out that during optimization for synthesis through IDT, an additional BsaI restriction site was added, which during Golden Gate assembly using BsaI, lead to problems.
Learn
The main learning outcome from the cloning cycle, was to always double and triple check your sequences before ordering. We decided to continue with our ten remaining laccase constructs.
Cycle B - First expression cycle
Design
We planned to produce our laccases in 15 mL YPD-NTC media using 100 mL shake flasks which would be incubated for about 72 hours.
Build
Media was prepared, shake flasks were filled and inoculated with a little dab of one colony . Shake flasks were then incubated at 29°C at 200 RPM over the weekend. 1 mL of each flask was centrifuged to only test activity in the supernatant, where the laccase should be due to the secretion factor.
Test
Laccase activity of the supernatant was measured with a spectrophotometer using an ABTS activity assay at pH 3, as these conditions were determined to work best by preliminary tests done on commercially available laccase. No measurable laccase activity could be detected.
Learn
Improvements were brainstormed, and we decided on three main differences to our first approach. We would start a preculture containing NTC from which cells would be taken, washed, and used to inoculate a production culture without NTC at a specific starting OD. Additional copper ions would be added as laccases are multicopper oxidases and need copper to function. The starting OD600 of our production culture would be much higher.
Cycle C – Second Expression Cycle
Design
A preculture would be started from a colony on a plate, grown for about 24 hours, subsequently the OD600 measured, and it was calculated what volume would be needed to get a starting OD600 of 8 for the production culture. The copper ion concentration we would add would be 50μM.
Build
We again prepared the media and inoculated straight prom the plate. After one day we had an OD600 of around forty in each flask. The correct volume of each flask was taken, washed with fresh YPD media, and used to inoculate the production cultures without antibiotic but this time containing additional copper.
Test
We tried to determine Laccase activity like before, but after initial spectrophotometric tests showed no activity, we decided to do a western blot of the supernatant and the pellet of cells. The western blot did not show any laccase, which explained the lack of laccase activity.
Learn
We will screen for other colonies, this time using primers which would bind to regions in the genome flanking the sites where the plasmid should integrate. This was done to rule out transient expression of cells only containing the plasmid, which would give them a temporary antibiotic resistance without integrating into the yeast genome. We might also increase the chances of protein expression using minimal medium such as Yeast Nitrogen Base (YNB).
Cycle D - Third Expression Cycle
Design
A new extensive round of colony PCR will be performed, screening eight colonies of each transformation for the integration of the plasmid into their genome. Moreover, we will use buffered YNB media at a pH of six and a copper concentration of 50µM for the production culture.
Build
After the colony PCR screening process using the new primers, new YPD-NTC precultures were inoculated with the promising candidates and left to incubate overnight. Meanwhile we prepared the YNB media which would be buffered using a phosphate buffer and again added copper ions to reach the desired concentration of 50μM. The next day, the precultures were harvested, washed, and again used to inoculate the production culture to an OD600 of 8.
Test
The same ABTS activity assay at pH 3 was performed and for the first time we had positive results showing laccase activity in the supernatant of our media. This activity could only be detected after extensive incubation of the assay which lead us to suspect that the concentration of laccase was very low. We then used a SpeedVac vacuum concentrator to reduce the volume of liquid and increase concentration of laccase. This was then used to run an SDS-PAGE which was then partly stained with Coomassie blue and partly using silver staining. Unfortunately, we were not yet able to detect the laccase proteins using SDS-PAGE. However, diclofenac concentration has been reduced by more than 40% in our samples analyzed by means of HPLC.
Learn
YNB media and the additional screening for colonies yielded positive results. To further improve the amount of expressed protein, switching to a promoter such as AOX1 might help, as an inducible expression system could be better suited to produce laccases. We had already started the cloning process, but due to time constraints we could not finish and test that theory. Also, improving detection of laccase, either through preceding purification using chromatography or using either laccase specific antibodies for western blotting or mass spectrometry would help to detect lower amounts of the enzyme.
Conclusion and Future Outlook
By following the DBTL cycle, we were able to successfully express and secrete functional laccase enzymes, as evidenced by measurable activity in the culture’s supernatant using the ABTS assay. Future work has to be done to determine if the TEV site and His-tag have a negative influence on laccase activity as we were only able to detect activity in strains which had the tags removed. Purification with chromatography and mass spectrometry detection of our enzymes would help to determine if the protein was expressed and just did not show any activity or if there was no laccase enzyme in the supernatant. Continuing the work on the inducible AOX1 promoter-based expression system might also hold a lot of potential for future iGEM teams.
Laccase Immobilization for Diclofenac/Ibuprofen Degradation
Overview
Our team focused on addressing the challenge of degrading pharmaceutical pollutants, such as Diclofenac and Ibuprofen, in wastewater using the fungal laccase Trametes versicolor. Initial experiments indicated that free laccase showed minimal activity at the neutral pH typical of wastewater, leading us to explore enzyme immobilization as a strategy to enhance its stability and performance. We utilized agar-agar as an immobilization matrix and intended to compare it with activated carbon; however, time constraints limited this comparison. The project aimed to determine the most effective method for enhancing laccase performance under wastewater conditions.
Cycle A – Preliminary Experiments
Design
The first step was to establish the optimal working conditions for Trametes versicolor laccase, particularly its pH and temperature stability. Our goal was to understand how these factors influenced the enzyme’s activity, especially under neutral pH conditions that resemble those in wastewater treatment plants.
Build
We conducted a series of experiments in which laccase was incubated in different pH buffers and temperatures. Enzymatic activity was assessed using ABTS as a substrate, with conversions monitored photometrically.
Test
The initial results suggested that laccase exhibited high activity at lower pH values but showed almost no activity at pH 7, which raised concerns about the viability of using free laccase directly in wastewater. However, further investigations revealed that the low activity at pH 7 was due to the limitations of ABTS as a substrate at neutral pH. When tested with other substrates, such as Diclofenac and Ibuprofen, laccase demonstrated activity even at pH 7, confirming that the enzyme can function effectively under neutral conditions with appropriate substrates. This insight underscores the importance of understanding the specific environmental conditions that influence enzymatic activity for effective bioremediation strategies.
Learn
Initially, we believed that T. versicolorlaccase had minimal activity at pH 7, leading us to focus on stabilizing the enzyme to make it functional in the neutral pH conditions of wastewater. This prompted us to explore immobilization strategies to enhance its stability and activity. Later, we discovered that free laccase could still function at pH 7 under specific conditions, so the pH issue was no longer a critical barrier. However, even though the pH problem was resolved, the practical challenges of using free laccase remained. Releasing free enzymes directly into wastewater would not be feasible or environmentally sustainable, as the enzyme could degrade or disperse without providing consistent treatment. This reinforced our decision to focus on immobilization as a necessary approach to ensure controlled and effective degradation of pharmaceutical pollutants.
Cycle B – Fe-Doped ZIF-8 Immobilization
Design
To improve laccase stability and catalytic efficiency, we planned to co-immobilize T. versicolorlaccase and the mediator ABTS in Fe-doped ZIF-8. This matrix was chosen for its ability to enhance enzyme stability, thermal resistance, and reusability, as well as its capacity to minimize secondary pollution caused by free ABTS.
Build
We aimed to co-immobilize laccase and ABTS using Fe-induced mineralization in a 1:1 ratio of Fe 2+to Zn2+, forming a stable ZIF-8 structure around the enzyme and mediator. This process would enhance laccase performance in degradation.
Test
We discovered that 2-Methylimidazole, a key component in ZIF-8 synthesis, had been listed by the ECHA as a substance of very high concern due to its reproductive toxicity. This regulatory issue prevented us from proceeding with the experiment.
Learn
The inability to complete the ZIF-8 immobilization highlighted the importance of considering regulatory restrictions in material selection. This experience emphasized that, beyond technical feasibility, we must always take environmental impact and the well-being of our communities into account when choosing materials and methods.
Cycle C – Agar-Agar-Immobilization
Design
Given the challenges faced in the preliminary experiments and ZIF-8 immobilization, we proceeded with agar-agar as a more accessible and safer immobilization matrix for laccase. Our objective was to enhance enzyme stability and reusability for pharmaceutical degradation, specifically targeting Diclofenac and Ibuprofen.
Build
We immobilized T. versicolorlaccase within agar-agar cubes and planned to test its degradation efficiency by exposing the immobilized enzyme to solutions containing Diclofenac and Ibuprofen.
Test
Using HPLC, we monitored the degradation of Diclofenac and Ibuprofen over a 4-day period. Immobilized laccase degraded 57% of Diclofenac at the higher enzyme concentration (0.60 mg/mL), outperforming the 48% achieved by free laccase. For Ibuprofen, degradation was similar between free and immobilized laccase, with both around 32% degradation.
We also compared the performance of reused and new immobilized laccase cubes. In this case, "reused cubes" refers to cubes that were washed multiple times after their first immobilization cycle before being used again in the next cycle. For Ibuprofen, both the reused and new cubes showed the same degradation efficiency, indicating that the immobilized enzyme retained its activity after reuse. This suggests that the immobilization method is suitable for long-term, cost-effective applications in wastewater treatment. Interestingly, for Diclofenac, the reused cubes performed better than the new ones, achieving 56% degradation within the first hour, likely due to variations in storage conditions that may have enhanced enzyme stability. This highlights the robustness of the immobilized laccase, particularly in its ability to be reused while maintaining or even improving degradation efficiency.
To confirm that laccase remained contained within the agar-agar cubes, we conducted enzyme inactivation experiments. After incubation, two aliquots were taken: one was directly analyzed by HPLC, and the other was heat-treated to inactivate any free laccase that may have leaked out before analysis. Both aliquots showed similar degradation results, confirming that no enzyme had leaked from the cubes during the process. This makes our immobilization method a strong candidate for sustainable and scalable wastewater treatment solutions.
Learn
Through our experiments, we confirmed that agar-agar is an effective immobilization matrix for T. versicolorlaccase, allowing for stable and reusable enzyme activity in the degradation of pharmaceutical pollutants. The enzyme retained its activity over multiple cycles, showing consistent degradation efficiency for both Diclofenac and Ibuprofen. Additionally, the enzyme inactivation experiments confirmed that the laccase remained securely contained within the agar-agar cubes, with no leakage into the surrounding environment. This ensures that our immobilization method offers both environmental safety and operational control.
We also learned that reused immobilized laccase cubes can sometimes outperform new ones, particularly for Diclofenac, potentially due to changes in storage conditions that may improve enzyme stability. This highlights the importance of monitoring storage parameters such as temperature and pH in future experiments to maintain or improve enzyme activity. Additionally, further use of the reused cubes over multiple cycles would be beneficial to fully assess their long-term efficiency and cost-effectiveness in practical wastewater treatment applications. Based on these findings, it would be beneficial to repeat the cycle with different laccase concentrations to further optimize degradation efficiency.
Cycle D – Activated Carbon Immobilization
Design
Following the success of agar-agar immobilization, we intended to explore activated carbon as another immobilization method. Activated carbon's high surface area and strong adsorptive properties were expected to support effective enzyme attachment and catalytic performance. We aimed to compare this method with the agar-agar approach to identify the most efficient immobilization strategy.
Build
We prepared activated carbon and attempted to immobilize laccase on its surface. However, during the process, we encountered difficulties filtering the fine activated carbon particles after treatment with HCl. Time constraints further prevented us from completing this method and making a direct comparison with agar-agar immobilization.
Test
Due to the challenges faced during preparation, we were unable to test the activated carbon immobilization method in time. As a result, no direct comparison with the agar-agar method was made.
Learn
Despite the setbacks, we gained valuable insights into the complexities of using activated carbon for enzyme immobilization. Future experiments could focus on optimizing the carbon treatment process and successfully completing the comparison with agar-agar to evaluate which immobilization technique is more effective for large-scale applications in wastewater treatment.
Conclusion
Through our engineering cycles, we confirmed that free laccase can function at neutral pH levels, such as those found in wastewater. However, we recognized that immobilization is essential for enhancing its stability and maintaining catalytic activity over time. The success of agar-agar immobilization demonstrates a promising solution for degrading pharmaceutical pollutants, with the added benefit of reusability.
Additionally, our enzyme inactivation experiments confirmed that laccase remained securely contained within the agar-agar cubes, with no leakage into the surroundings, ensuring controlled and environmentally safe application. Furthermore, reused immobilized laccase cubes retained or even exceeded the efficiency of new cubes, particularly for Diclofenac, likely due to storage effects, highlighting the potential for long-term, cost-effective wastewater treatment solutions.
Introduction and background
The biosensor project is inspired by previous publications, which showed that diclofenac may interfere with certain cellular pathways in S. cerevisiae, eventually leading to upregulation of genes, notably PDR5 (Pleiotropic Drug Resistance 5), through promoter activation [1]. PDR5 gene encodes an ABC transporter. One key transcription factor that activates PDR5 expression in response to diclofenac treatment is pdr1p/pdr3p heterodimer. Schuller and colleagues[1] have shown previously that modifications of the PDR5 promoters, essentially increasing copy numbers of the pdr1p/pdr3p binding consensus sequences increases sensitivity of the promoter response to diclofenac, measured by fluorescent reporter gene assay. Further characterisations of additional promoters containing pdr1p and pdr3p binding sites[2] encouraged us to include more promoter candidates to test whether and to what extend do they respond to diclofenac.
As for ibuprofen, to our knowledge, current challenge has it that there is no known direct and native pathway in S. cerevisiae that can be modified and utilised for our biosensor project. Previous genome-wide deletion screening against chemical and oxidative sensitivity has shown knockout of various candidate genes confer susceptibility to ibuprofen[3], implying existing coping mechanism(s) that underlie fitness of WT cells under ibuprofen treatment. Furthermore, He and colleagues[4] have elucidated that ibuprofen induces degradation of high affinity aromatic amino acid transporter tat2p; under ibuprofen treatment, cells are deprived of aromatic amino acids, particularly tryptophan. Thus, we are driven to look for genes that are potentially upregulated under low tryptophan level to test our hypothesis whether such promoters will also be upregulated with the presence of ibuprofen. We selected 4 promoters that could potentially respond to ibuprofen, namely pPYK1, pCYS3, pSHM1, and pHIS4[5].
Design
Reporter Assay
To assess whether or to what extend these promoters respond to the NSAIDs, we designed reporter gene essay, namely fusing various promoters with the downstream reporter gene (sfGFP). By comparing levels of fluorescence emission among different cells transformed with various promoter-sfGFP constructs, we might be able to observe and compare the dynamic ranges of these biosensors across various drug concentrations (Fig.1).
Gibson Assembly
We use Gibson assembly as a restriction enzyme-independent cloning strategy to make our constructs due to the fact that some of our selected promoters contain BbsI and BsaI cutting sites. These might affect Golden Gate Cloning efficiency. Our cloning strategy for Biosensor sub-project consists of promoter isolation and addition of homology sequencing to DNA fragments by extended primer (overhang primer) PCR (Fig.2).
Engineering success and next steps
Despite that we only obtained one single colony of one construct which showed positive after colony PCR at the point of Wiki-freeze (Fig.3a), we have gone through at least one design, build, test, and learn cycle. The Sanger sequencing further validated our assembly (Fig.3b). We firstly designed four-fragment Gibson assembly reactions to bring together our genetic and regulatory elements to the goal plasmid; we executed our subsequent experiments to build and acquire our basic parts for our Gibson assembly to work. In these processes, we went through multiple failures and additional trouble-shooting steps, such as the promoter amplification inefficiency and determining best annealing temperature for our promoter isolation PCR by thermogradient PCR. Without these steps, we would never have learnt from a practical perspective that a four-part Gibson assembly reaction seemed to be very inefficient in our current settings, given that a) We observed an increase in transformation efficiency when we switched from heat-shock to electroporation; and b) We only obtained one single positive colony. Through these small intermediate conclusions, we are inspired to further change and test with different parameters. From current results, we believe that it would be meaningful to a) Further increase the reaction concentration of Gibson assembly; b) Test new Gibson compatible commercial kits; and c) Consulting experts (such as Janoš) for advices and experiences. From a bigger picture, we believe it is worthy to try additional promoters from previous study[5] that responded to Ibuprofen.
In the earlier phase of the Sub-project, we have mistakenly matched the wrong name to the gene number denoted in the expression heat map (see Liu et al. 2021[5]). As a result, only 3 out of 6 selected promoters will potentially respond to ibuprofen and diclofenac treatment. Thus, we will also include addition promoters which potentially respond to ibuprofen and our current knowledge about them in our basic part contribution entry. It would be absolutely inspiring and proud to see future generation iGEMers to continue our journey to design, build, and test their biosensor systems that detect micropollutants, and learn from their own mistakes, experiences, and successes.
As for our next step in the biosensor project, we will continue with our trouble shooting and optimisation for Gibson Assembly. Transformation in S. cerevisiae with various concentrations of diclofenac with negative control (transformation with backbone plasmid) will further elucidate whether at all will this promoter indeed or to what extent will it respond to diclofenac. We are looking forward to sharing these exciting results with judges at the Grand Jamboree 2024 in Paris.
References
- Schuller, A., Rödel, G. & Ostermann, K. Tuning the Sensitivity of the PDR5 Promoter-Based Detection of Diclofenac in Yeast Biosensors. Sensors 17, 1506 (2017).
- Buechel, E. R. & Pinkett, H. W. Activity of the pleiotropic drug resistance transcription factors Pdr1p and Pdr3p is modulated by binding site flanking sequences. FEBS Lett 598, 169–186 (2024).
- Tucker, C. L. & Fields, S. Quantitative genome-wide analysis of yeast deletion strain sensitivities to oxidative and chemical stress. Comp Funct Genomics 5, 216–224 (2004).
- He, C. et al. Enhanced Longevity by Ibuprofen, Conserved in Multiple Species, Occurs in Yeast through Inhibition of Tryptophan Import. PLoS Genet 10, e1004860 (2014).
- Liu, H.-L., Wang, C. H.-T., Chiang, E.-P. I., Huang, C.-C. & Li, W.-H. Tryptophan plays an important role in yeast’s tolerance to isobutanol. Biotechnol Biofuels 14, 200 (2021).