Team:Stockholm/Demonstration

Demonstration

After spending time investigating what problems can be solved through synthetic biology, we landed on the track of helping wastewater treatment facilities solve the issue of degrading pharmaceutical residues in wastewater before they reach the environment. As a solution, we have developed an innovative enzymatic approach towards tackling this problem. We built a method incorporating a model that can evolve from experimental analysis to improve the specificity of a fungal laccase towards a specific antibiotic. As a proof of concept, we have demonstrated our work with sulfamethoxazole – one of the persistent antibiotics found in the Baltic Sea region. We then progressed out of the lab and designed a prototype to demonstrate how this methodology can be implemented in wastewater treatment plants. A huge contributing factor to the design was our many visits to wastewater treatment facilities and companies within the field to investigate our implementation.

Can Laccases Reduce the Toxicity of Sulfamethoxazole (SMX)?

Before we go further with our concept, it is important to prove that laccases can degrade SMX to products that are non-toxic and are hence safe for the environment. Through ecotoxicity testing with a commercial laccase and the wild type fungal laccase that we produced, we were able to conclude that SMX ecotoxicity was reduced and that the reaction products are safe.

An Iterative Evolutionary Approach Towards Enzyme Engineering

As elaborated on our Project Design page, the main objective of engineering the wild type fungal laccase was to increase its specificity towards SMX to improve its degradation rate. Multiple engineering strategies can be performed and we have explained why we specifically chose rational enzyme design for this project. We have also chosen Pichia pastoris as our production platform to express the engineered enzymes. Ultimately, the project went through multiple phases:

1. Modeling Reaction Mechanism

The first phase of rational enzyme design was to find the reaction mechanism for SMX with laccases and the parameters that can describe it. As part of publishable work, we were able to model the reaction mechanism of our enzyme.

2. Validating the Model

The second phase was to understand and learn about our system starting with creating a valid model for the wild type laccase. Through our simulations we were able to determine how the laccase behaved in solution and how it interacted with a given substrate. We then inferred under which conditions the system operates and thereafter designed experiments that can directly prove or disprove our claims from the model. This was the first cycle of our design, build, test methodology:

• Design: From the model we were able to calculate theoretical values of the catalytic parameter kcat.
• Build: We were successful in expressing, producing, and purifying the wild type laccase from Pichia.
• Test: An activity assay with the model-substrate ABTS was conducted to calculate experimental Kcat.

As a result, we succeeded in validating our model as the experimental Kcat value confirmed the theoretical value of kcat for the model-substrate ABTS. This also standardized our pipeline with assays development for analysis, where mutants can be similarly tested for validation and then compared with the wild type laccase. (More can be found in our Model Page.)

3. Creating a Mutant

This phase describes the evolutionary iteration of our design, build, test methodology towards creating successful mutants.

From our first simulation of the mutated enzyme the design focused on increasing the catalytic activity of degrading SMX through making it fit better into the active site. We then successfully transformed the selected mutant into P. pastoris. However, in the testing phase the enzyme was inactive and we inferred it could be due to misfolding. This in turn caused us to iterate back into the design phase to test this assumption. As a result, this gave us a better insight on the folding modes of our simulated protein as our assumption was confirmed. Thus, this helped us to achieve our objective by expanding our model and introducing another generation of mutations, where the modeled kcat was 10 times better than the wild type kcat. In the future, the cycle can be continued to express and test the next generation of mutants.

Product Design & Real Life Implementation

For the engineered laccase to be introduced at Wastewater Treatment Plants (WWTPs) there needs to be a way for them to be captured and reused. Therefore to make this happen, we developed a smart strategy of immobilizing them to magnetic beads so that they can be recovered via magnetism.

1- Laccases immobilized on magnetic beads were proven to be functional in the ABTS activity assay:

Figure 2A. Immobilized magnetic beads showing activity when ABTS was added. Figure 2B. Recovery Process in Lab Scale

2- After utilizing advice from experts, the following implementation strategy for large scale recovery was structured:

Figure 3. A detailed outline of our prototype showing how it can be implemented in WWTPs.