Background

Endocrine disruptors (EDCs) are low molecular weight compounds that can interfere with hormone activity in living organisms. They are capable of causing several dysfunctions, such as reduced fertility and reproductive diseases such as endometrial or testicular cancer [1, 2] . 17α-ethinylestradiol (EE2) is an EDC which mimics its natural analogue 17β-estradiol (E2), and is one of the most used medications for humans (for birth control pills) and livestock manure (to promote growth). Thus, EE2 has been found in many ecological communities since its excretion through urine, and further discharge into the sewage led to its accumulation in wastewater treatment facilities (WWTF) that supplies local water distribution systems, which provides water for human and animal consumption, agricultural irrigation systems and in some cases, are discharged into aquatic ecosystems. The inability of WWTF to remove EE2 has become a severe problem worldwide in urbanized regions due to its high resistance to degradation and its bioaccumulation capacity, as well as its capacity to have detrimental effects at low concentrations [3, 4] . According to literature, the most reported effects of EE2 are involved in cancer development [5] as well as in impairing fish reproduction through demasculinization and disruption of the endocrine system [6, 7], altering both human health and ecosystem dynamics.

In Nuevo León, Mexico, the water crisis has prompted the government to explore indirect water reuse as a solution [8]. However, the presence of EE2 in the wastewater treatment plant effluent in Monterrey poses a significant risk. After the disinfection phase in the plant, EE2 levels averaging 9.8 ng/L are 281 times higher than the acceptable standard of 0.035 ng/L [9, 10]. Conventional treatments that could prevent these high levels like nitrifying activated sludge (NAS) are insufficient, and advanced methods like chemical removal and activated carbon are costly [11]. A promising solution involving low-cost materials such as algae and fungi, may offer economic and operational benefits without producing harmful by-products [12] . Additionally, using genetically modified organisms (GMOs) capable to intake and metabolize these compounds could enhance their removal from wastewater [13] .

The ALIVE project seeks to selectively degrade EE2 through a genetically modified algae that activates the synthesis of laccase in the presence of the component, thus preventing the rise of its toxic levels.

Laccase

Laccases, particularly those from the fungus Trametes versicolor> (Tvlac), are multi-copper oxidases known for their ability to catalyze the oxidation of a broad range of organic compounds, including phenolic and aromatic substrates [14] One of the key applications of Tvlac lies in its capacity to inactivate estrogenic pollutants, such as 17α-ethinylestradiol (EE2), making it a promising candidate for addressing environmental contaminants [15].

The mechanism by which laccase acts on EE2 involves an electron transfer process at the enzyme's type-1 copper (T1-Cu) site. This catalyzes the one-electron oxidation of EE2, generating reactive estrogen radical intermediates. These radicals can undergo oxidative coupling, forming polymeric products through carbon-carbon and carbon-oxygen covalent linkages, which results in oligomers of EE2 [16]. In some cases, the radicals self-polymerize, creating dimeric and oligomeric species characterized by aromatic structures and phenolic -OH groups. As the reaction progresses, the polymeric products that form can obscure the reactive sites of EE2, thus decreasing the efficiency of further oxidation reactions [17] . This process effectively sequesters the EE2 compounds within larger macromolecular structures, a transformation that aligns with natural detoxification pathways. By polymerizing, environmental estrogens become less bioavailable and significantly less toxic to aquatic organisms and humans. Studies have demonstrated that the biotoxicity of these compounds is greatly diminished following their conversion into oligomers [16].

Despite these advantages, the application of Tvlac in environmental remediation faces several challenges. Laccase activity is highly sensitive to fluctuations in pH and temperature, which can limit its functionality in natural environments where these parameters are not constant [18] . Additionally, laccase tends to interact with humic acids and other naturally occurring small phenolic compounds, which are also secreted by the Trametes versicolor fungus [17]. These interactions can interfere with the enzyme's efficiency in oxidizing target substrates like EE2, reducing its overall conversion efficiency. Consequently, while Tvlac shows great potential for inactivating estrogenic pollutants, these operational limitations must be addressed to optimize its effectiveness in real-world environmental conditions.

Chlorella vulgaris

The second part of our project focuses on addressing the limitations of Trametes versicolor laccase production by utilizing Chlorella vulgaris as the host organism for laccase synthesis. C. vulgaris, a versatile microalga commonly found in freshwater, marine, and terrestrial environments, does not naturally produce laccase like T. versicolor. However, it possesses several advantageous traits that make it a suitable candidate for large-scale enzyme production. With its high photosynthetic efficiency and rapid growth, C. vulgaris can thrive under diverse conditions, including autotrophic, mixotrophic, and heterotrophic modes, making it highly adaptable to various environments [19].

One challenge we face is regulating the expression of the TvLac genes in the transformed algae, ensuring that laccase production occurs only in the presence of 17α-ethinylestradiol (EE2) in the environment, since overproduction of laccase could lead to C. vulgaris cell death [20]. Overcoming this issue is key to optimizing the efficiency and specificity of the system.

ESTER

We designed a protein named ESTER (Estrogen-Sensitive Transcriptional Element Regulator) based on the findings of Ibero et al. (2021) [21] , who identified a TetR-like protein in Caenibius tardaugens that regulates the transcription of genes involved in the degradation of estradiol and testosterone.

However, the primary limitation of using this protein for the detection of EE2 is lack of affinity for this contaminant. To address this, we performed directed mutagenesis on the protein using advanced simulation software to identify key amino acids involved in its interaction with EE2 and to predict the effects of these mutations on the protein's function. Our goal was to enhance its binding affinity for this contaminant. Additionally, we modified the protein to alter its binding sequence to a specific DNA sequence based on the tetracycline repressor DNA binding domain [22, 23] .The mutations introduced to the protein were designed to enhance its specificity for EE2 and to target its binding to a specific DNA sequence. This modified protein will bind to DNA and inhibit the transcription of downstream genes by obstructing RNA polymerase. In the presence of EE2, the compound will bind to our dimeric protein, reducing its affinity for DNA. Consequently, the protein will release the DNA, allowing transcription of the genes to proceed.

Some of the softwares used are Alphafold3 [24] for 3D modeling, Autodock Vina [25] and Chimera [26] for molecular docking, Rosetta [27] for the refinement of dockings, Discovery studio [28] to analyze protein-ligand interactions and Gromacs [29] to simulate molecular dynamics.

References

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