Phthalates are a group of chemical compounds that are used in industry as plasticisers to improve their flexibility, transparency, and durability. Their derivatives do not bind permanently to polymers, so they are easily released into the environment and, as endocrine active substances, adversely affect the endocrine and reproductive systems of living organisms. In our project we aim to use synthetic biology to fight with phthalic ester contamination of water by designing and manufacturing a prototype device containing a biological filter with immobilized enzymes capable of degrading phthalates. Our goal is to develop a new system using a genetic engineering tools, which will incorporate a fusion of the cellulose binding module, cohesins, dockerins, and a pair of enzymes. Working harmoniously, these components will perform a two-step enzymatic reaction that effectively breaks down phthalates into significantly safer compounds.
Phthalates are a group of chemical compounds that pose a threat to the proper functioning of the endocrine system in humans and animals. As they are still being researched and their danger is slowly being realized, governments and institutions have only began enforcing bans on their industrial usage. In March 2018, European Union has updated its restrictions on industry phthalate use, limiting the types of materials which can contain these harmful chemicals. This change was caused by ongoing research on the effects of long-term exposure to phthalates on humans and animals. Although the restrictions have helped shed a light on the issue, they are still far from ideal. Notably, the regulations omit food contact materials, which are the largest source of phthalate exposure for humans. Phthalates have a negative impact on the male reproductive system in animals, causing hypospadias, cryptorchidism, lower levels of testosterone, and lower sperm counts [1]. It is proposed that phthalates is the reason for the global decline in the diversity of reptile species. It interferes with the growth of internal reproductive organs in animals, even at very low concentrations, causing underdevelopment of the vas deferens, vacuolization of Sertoli cell cytoplasm, lymphocyte infiltration, and gonadal dysgenesis [2].
We want to take a step further and not only educate on this issue, but also try to find a scientific solution to minimizing phthalic acid esters exposure in everyday life. As of 2023, a biological device able to degrade phthalic acid esters has not yet been created, which makes our project revolutionary in the field of bioremediation. Carrying out at least some of the planned experiments will result in the first practical testing of the effectiveness of our chosen enzymes, which will be a milestone on the road to developing methods of purifying water from phthalates. We will then go on to develop and build a working 3D model of our water filtering device.
The proximity of the Baltic Sea has inspired us to focus on local issues connected with marine organisms and seabed pollution. In 2019, our University took part in The NonHazCity project which resulted in detailed analysis of presence of hazardous chemicals in sewage water samples taken in several cities located at the coast of the Baltic Sea, including Gdańsk. The research has shown that there was a 100% frequency of phthalate detection in sewage water samples from Gdańsk, with DBP being detected in 84% of the samples. These numbers picture just how serious the exposure to phthalates can be and how little is currently done to limit it. The sewage water eventually flows into the Baltic Sea, whose already endangered fauna becomes exposed to various endocrine disrupting chemicals present in wastewater. We wanted to find a way to safely filter water, without using GMOs, and in this way limit the concentration on pthtalic acid esters present in the environment. In addition, Gdansk is one of the largest tourist cities, where millions of people come every year to enjoy the beautiful sandy beaches and warm water. A method of purifying wastewater from phthalates could be implemented in Gdansk's wastewater treatment plant, which would have a positive impact on the quality of the sea water in the bay and tap water. Clean tap water is becoming increasingly important as more and more people in Poland give up bottled water for tap water.
Phthalates are used as an additive in plastic products all over the world, so we believe that water contamination is not only in the Baltic Sea, but also in other waters, as future analysis should show. It is worth mentioning the The Great Pacific garbage patch, which consists largely of plastic waste that will float in the water for many years and degrade over time.
One solution to environmental pollution with phthalates, using synthetic biology, was proposed by Dinget al. [3]. They developed a transgenic Escherichia coli which displayed a carboxylesterase CarEW from Bacillus sp. K91 on its surface, that could be used as a whole-cell biocatalyst for phthalate esters degradation. CarEW is catalyzing the reaction of hydrolysis of ester bonds in diisobutyl phthalate (DiBP) producing monoisobutyl phthalate, isobutanol and phthalic acid. It was anchored to the cell outer membrane by fusion with truncated N-terminal domain of ice nucleation protein from Pseudomonas syringae acting as anchoring motif. It was proven, that this modification had no negative effect on cell growth or membrane integrity. DiBP degradation rate of this whole cell catalyst was comparable to purified enzyme.
We are planning to use these previous achievements made by iGEM teams mentioned above to improve our own device which contains an immobilized enzymatic complex also based on cellulosome. The data from other iGEM teams is going to help us advance our own construct, which composes of cellulose biding module, cohesins, dockerins and two enzymes responsible for degradation of phthalates. It will not only help us immobilize enzymes on cellulose, which enhances their activity, but will also allow us to perform a two step enzymatic reaction that degrades phthalates to much less hazardous substances. In addition, our final product will not contain any living GMO in view of the fact that the aim of our project is to use immobilized enzymatic complex, not immobilized bacteria, in contrary to iGEM teams mentioned before.
Cellulosomes are large protein complexes found on the surface of many cellulose-degrading microorganisms [4]. Their main components are cohesin domains (cohesins), which, arranged side by side, form a protein scaffold for cellulolytic enzymes. These enzymes have an embedded dockerin domain (dockerin), which has a high affinity for a particular type of cohesin. Thus, these enzymes can form a complex with cohesins to form a functional cellulosome as a result. In addition, each cellulosome has a cellulose binding domain (CBM), which strongly binds the entire complex to cellulose. Thanks to the immobilization and the mutual proximity of the enzymes, cellulose degradation is much more efficient.
Figure 1. Graphic representation of our enzyme complex immobilized on cellulose.
Our design consists of a cellulose binding module, cohesins, dockerins, and two phthalate-degrading enzymes. It has been already reported that immobilization enhance enzyme activity, our approach allows for two step enzymatic reaction that degrades phthalates. To carry out this process, we will use enzymes of two classes - an esterase and a decarboxylase. Enzymes from the esterase group catalyze the hydrolysis reaction of ester bonds, while decarboxylases allow the detachment of the carboxyl group. In the literature, we found examples of enzymes that carry out the degradation of phthalates, including DBP. We are keen to achieve the highest efficiency of phthalate degradation, so we decided to immobilize the selected enzymes to improve the efficiency of the catalyzed reactions [5]. We will use a synthetic mini-cellulosome as the immobilization platform.
Figure 2. Scheme of enzymatic degradation of phthalates.
We hope that our laboratory results will contribute to the discovery of a metabolic pathway that allows enzymatic degradation of phthalates and inspire other research teams to use cellulosome-based systems. We hope that the solution we have developed will be feasible for use in wastewater treatment plants and have a positive, long-term impact on community health, and will be widely used in other water treatment units in the future.
