Endocrine disrupting chemicals (EDCs) are exogenous compounds known to cause disruption in the development and fertility of living organisms. These hormonally active agents enter the environment through various mechanisms of air, water, and soil contamination. Natural and synthetic estrogens are some of the most potent endocrine disrupting chemicals found in municipal wastewater and effluent which enters aquatic ecosystems. The synthetic estrogen 17α-ethynylestradiol (EE2), a compound found mainly in contraceptives such as birth control pills, is the most ecotoxic and difficult to degrade due to the presence of a sterically hindering alkyne group. Levels as low as 4 ng/L in water have been recorded to cause the development of secondary sex characteristics in male specimens of the fathead minnow (Pimephales promelas), with a lack of any sexual differentiation at higher concentrations. Prevalence of EE2 in aquatic bionetworks is exacerbated by runoff from anthropogenic sources.
Research revolving around biotechnological applications in bioremediation, such as the usage of microbes and enzymes, is ongoing. Laccases are a prominent class of multi-copper oxidases (MCOs) found in species of fungi and bacteria. They are versatile enzymes that are proven to be capable of degrading phenolic compounds and this makes their implementation in industrial and municipal wastewater treatment attractive.
Wastewater treatment plants (WWTPs) are the primary barrier in preventing toxin secretion into the environment. Sorption and biodegradation are commonly utilized methods in the removal of organic pollutants from municipal wastewater, including estrogens. However, the reduction of estrogenic compounds is often incomplete and this results in the final wastewater effluent containing trace amounts of EE2 that are released into aquatic environments. Detecting lower levels of EE2 is not equivalent to decreased ecotoxicity, therefore synthetic estrogen pollution remains a problem.
Why synthetic biology?
When tackling an issue with so many gaps in knowledge in current literature, flexibility in the approach you take is ideal. Synthetic biology provides this necessary adaptability and so much more through allowing us to combine nature and technology to solve novel problems. With EE2, we thought that looking at natural laccases may provide us with a solution. By engineering plasmid into an E.coli bacterium, we would be able to produce a high concentration of our laccase while potentially avoiding the risks coming with using other chemicals to degrade EE2. By creating our own plasmid, we can also incorporate other genes of interest such as signal peptide like NSP4 in order to further optimize the production. Furthermore, our bacteria could then be added to the bacterial treatment section of wastewater plants, further improving an existing system.
Why Bacillus licheniformis? (Our solution)
This project aims to use Esherichia coli as a vector for expression and production of a bacterial laccase that will degrade EE2 at an optimal wastewater pH (6-8). The project seeks to develop recombinant E.coli that is able to withstand wastewater matrix conditions and maintain high activity and increased efficiency. It is important to not increase the ecotoxicity from the final wastewater effluent so this project also aims to decrease the production of harmful secondary products. Additionally, for the consideration of biosafety controls, this project seeks to implement a killswitch system.
References
Listed below are some of the main references that were referred back to during project development and were used for general background knowledge and laccase considerations.
Aris, Ahmad Zaharin, et al. “Occurrence of 17α-Ethynylestradiol (EE2) in the Environment and Effect on Exposed Biota: A Review.” Environment International, vol. 69, 2014, pp. 104–19, https://doi.org/10.1016/j.envint.2014.04.011.
Arregui, Leticia, et al. “Laccases: Structure, Function, and Potential Application in Water Bioremediation.” Microbial Cell Factories, vol. 18, no. 1, 2019, p. 200, https://doi.org/10.1186/s12934-019-1248-0.
Gupta, Nirupama, and Edgardo T. Farinas. “Directed Evolution of CotA Laccase for Increased Substrate Specificity Using Bacillus Subtilis Spores.” Protein Engineering, Design and Selection, vol 23, no. 8, 2010, pp. 679–82, https://doi.org/10.1093/protein/gzq036.
Falade, Ayodeji O., et al. “Ligninolytic Enzymes: Versatile Biocatalysts for the Elimination of Endocrine‐disrupting Chemicals in Wastewater.” MicrobiologyOpen, vol. 7, no. 6, 2018, p. e00722, https://doi.org/10.1002/mbo3.722.
Janusz, Grzegorz, et al. “Laccase Properties, Physiological Functions, and Evolution.” International Journal of Molecular Sciences, vol. 21, no. 3, Jan. 2020, p. 966, https://doi.org/10.3390/ijms21030966.
Ihssen, Julian, et al. “Biochemical Properties and Yields of Diverse Bacterial Laccase-like Multicopper Oxidases Expressed in Escherichia Coli.” Scientific Reports, vol. 5, no. 1, 2015, p. 10465, https://doi.org/10.1038/srep10465.
Racz, LeeAnn, and Ramesh K. Goel. “Fate and Removal of Estrogens in Municipal Wastewater.” J. Environ. Monit., vol. 12, no. 1, 2010, pp. 58–70, https://doi.org/10.1039/B917298J.
Spina, Federica, et al. “Ecofriendly Laccases Treatment to Challenge Micropollutants Issue in Municipal Wastewaters.” Environmental Pollution, vol. 257, 2020, p. 113579, https://doi.org/10.1016/j.envpol.2019.113579.