Description
Project inspiration
“Water is life’s matter and matrix, mother and medium. There is no life without water”1
Liquid water is and was a central element in the origin and development of life on earth and has not lost its importance to this day.2 As the essence of life, it is needed by every living thing. Clean water ensures survival and the species. However, water is not only a vital resource, but also a habitat in its own right, occupying a dominant role in our biosphere due to its enormous size. Aquatic ecosystems contain not only plants and algae, but also animals and insects of all kinds that depend on the integrity of their habitat. Likewise, these ecosystems provide an elementary service to us humans both in terms of nutrition and hygiene.3 The Social Development Goals for 2030 include protecting our environment, specifically protecting life underwater and on land. 4 To protect the oceans, the current threats to them must be stopped. Above all, pollution of the oceans must be stopped to ensure healthy and biodiverse marine and coastal ecosystems. Land ecosystems are also increasingly disturbed, resulting in environmental disasters and threats to our climate.5 It is time to give back to our environment by addressing the mistakes of past decades and trying to undo them. What is striking is the increased occurrence of sudden species extinctions and changes in ecosystems in recent years and decades. Climate change has a major influence here, of course, but the pollution of our waters is also repeatedly a cause of change and destruction of our environment. With the destruction of the environment, man signs his own death warrant by taking away the basis of his life with his actions. Because everyone depends on the environment as a good, without it we and also all other living beings cannot survive. The poisoning of the soil, the extinction of species of animals and plants and contaminated, unhealthy food are only a few consequences of environmental destruction.6
The Problem - Water Pollution
Waste, plastic, oil
Despite its importance, water is polluted in many different ways. All types of pollution pollute the environment and destroy important ecosystems. In addition to the pollution of water by waste, mainly plastics, and oils, which is present in the media, there is an even greater but invisible threat.
Chemical toxins (pesticides, medical waste)
Due to the continued growth of the global population, the demand for food and the threat of pathogens is continuously increasing. To meet the demand for food, fertilizers, as well as pesticides, are used to protect crops and safeguard the harvest. These pesticides, such as epoxiconazole, are often insoluble in water 7 but can still be carried away from their intended use by environmental factors such as rain or wind. Drug residues from antibiotics or painkillers, such as diclofenac, can also enter the environment through the wastewater system and inadequate treatment, harming naturally occurring bacterial populations or marine life.8
Wastewater treatment
Wastewater treatment plants use physical methods as well as biological and chemical processes to remove pollutants from wastewater. Even though these processes are designed to work with the highest possible efficiency, small wastewater treatment plants in particular cannot profitably use all of the common processes, such as ozone gas purification and activated carbon filters. Due to the lack of purification stages, there is a possibility that not all of the contaminants, especially chemical waste, will be removed from the water. The substances that cannot be removed by the treatment processes thus accumulate in the environment and can become a chronic burden for the aquatic and terrestrial organisms concerned. Furthermore, the continuous use of the activated carbon column is associated with a high labor input and a negative CO2 balance. The activated carbon must be cleaned by pyrolysis after prolonged use, this process is energy-intensive and material from the column is lost during each cleaning process. 9,10
As chemicals like insecticides and herbicides as well as pharmaceuticals cannot often be broken down by biological and chemical processes, these advanced cleaning methods often are located as optional stages in the last parts of the wastewater treatment cycle. Here we want to use our genetically engineered algae as a biological stage in wastewater treatment to break down these previously hard to get rid of chemicals in a sustainable and eco-friendly, carbon-neutral fashion. Since Chlamydomonas can grow photosynthetically, no additional growth supplements or expensive growth conditions would have to be established.
Much of the insecticides and herbicides applied to fields on farms leach, carried away by rain or wind, into bodies of water and deeper layers of soil where these contaminants can pollute groundwater. By not breaking down these chemicals in a treatment plant before the water is used to produce food or other industrial processes, the creatures and people who come into contact with this water can be chronically exposed to pesticides and drug residues. This chronic exposure to foreign substances, such as the hormonal drug residue estradiol or the antibiotic erythromycin, can negatively impact the health of all affected (micro)organisms. Estradiol, for example, causes reproductive deficits when chronically exposed, but also hypertension and anxiety-disorder-like behavior.11 Erythromycin, a commonly used antibiotic causes defects in the photosynthetic mechanisms of prokaryotic and eukaryotic organisms when chronically exposed to it.12
Our solution – CYPurify
Our project
Our vision is to detoxify the toxins present in water, such as herbicides, insecticides and medical waste. We would like to accomplish this through bioremediation. For this purpose, we modify the unicellular green alga Chlamydomonas reinhardtii with the so-called cytochrome P450 enzymes (CYP enzymes). We want to find and implement new CYP enzymes to target specific pollutants around the world. We want to show that we have the tools to solve a fundamental environmental crisis that affects each and every one of us. The goal of our project is to reverse decades of poisoning our environment and help nature regenerate.
Cytochrome P450 enzymes
Cytochrome P450 enzymes are a large superenzyme family of heme-containing monooxygenases and are found in all domains of life, giving them incredible diversity.13 They play a major role in xenobiotic metabolism and are essential for the oxidative biotransformation of pharmaceuticals, especially in humans. However, they also play an important role in the synthesis of various endogenous substances, such as steroid hormones, bile acid, etc.14 These enzymes are mainly localized in the liver, but also in the luge, kidney and intestine. In terms of pharmacology, CYP enzymes have been very well researched and also used for drug development. CYP enzymes show enormous potential for bioremediation, which, however, currently remains unexploited. However, their complexity should not be underestimated, which has also made their use difficult up to now. These enzymes are membrane-bound and very reactive. Although their broad substrate spectrum enables a large number of reactions, these enzymes can also lead to toxification with certain substrates. Overexpression of CYP enzymes in other organisms mediates various resistances to pesticides or herbicides. 15 Currently, CYP enzymes are not commonly used in synthetic biology in the context of bioremediation, but rather to enable the synthesis of high-value products such as the precursor of the antimalarial drug artemisinic acid in genetically modified yeast.
We chose to develop constructs for the enzymes CYP2D6, CYP3A4, CamC, CYP9Q3 and CYP81A10v7.
We chose to analyse the humane CYP2D6 and CYP3A4 as they are responsible for metabolizing almost all pharmaceutical compounds humans are exposed to on a day-to-day basis.
CamC is a soluble bacterial CYP-enzyme capable of breaking down polyhalogenated hydrocarbons. This raised our curiosity, moving us to develop constructs for this enzyme as well.
CYP9Q3 has its origins in the common bee, Apis mellifera and is responsible for the degradation of neonicotinoid insecticides. 16
This catalytic potential seemed wonderful for our project, as we are targeting insecticide contamination in wastewater among other things.
CYP81A10v7 is an enzyme found in resistant strains of the grass Lolium rigidum. It can break down most known classes of herbicides, such as acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides as well as hydroxyphenylpyruvate dioxygenase-inhibiting herbicides, photosystem II-inhibiting herbicides and the tubulin-inhibiting herbicide trifluralin.17 Since this enzyme is capable of breaking down such a vast number of herbicides, we definitely wanted to create a construct for Chlamy to give it the ability to break down these toxins in the environment.
Chlamydomonas reinhardtii
Chlamydomonas reinhardtii is a eukaryotic unicellular green alga and is suitable for research and use in biotechnology due to its short generation time and inexpensive cultivation. This green alga has a small, haploid genome and organelles typical of plants, such as chloroplasts, cell walls and vacuoles. The alga is able to grow photoheterotrophically with photosynthesis or in the absence of light, acetate as the sole source of energy and carbon. Chlamydomonas also produces NADPH, reduction equivalents required by P450 enzymes for activity, during photosynthesis. Chlamydomonas is a well-studied organism that is easy to modify genetically. It is suitable in biotechnology research and as a good precursor organism to higher plants because it has no tissues and few isoforms of proteins.
Modular Cloning
The Modular Cloning System (MoCLo) is based on Golden Gate Cloning. The use of standardized fusion sites, generated by TypIIS restriction enzymes, allows an easy and fast assembly of the parts. The special feature of these restriction enzymes is that they cut outside their recognition sequence. For the ligation of the individual DNA fragments, all required parts can be added to a single reaction mix together with the T4 DNA ligase and the TypIIS restriction enzyme. However, MoClo also requires the prior generation of standardized modules that have been both codon-optimized for the host and cleared of unwanted TypeIIS restriction sites. Another advantage here is that this method has already been optimized for Chlamydomonas reinhardtii. 16
- Wastewater analysis and drugs - a European cross-city study.
Article - Szent-Györgyi A. Biology and pathology of water. Perspect Biol Med. 1971;14:239–249.
- Ball P. Water is an active matrix of life for cell and molecular biology. Proc Natl Acad Sci USA. 2017;114:13327–13335.
- Wasser & Ökologie - Helmholtz-Zentrum für Umweltforschung UFZ
Article - THE 17 GOALS | Sustainable Development (un.org)
Article - Agenda 2030: Unsere Nachhaltigkeitsziele | Bundesregierung
Article - Ursachen und Folgen von Umweltproblemen
Article - http://www.epa.gov/opprd001/factsheets/epoxiconazole.pdf
pdf - Chronic exposure to diclofenac on two freshwater cladocerans and Japanese medaka, Ecotoxicology and Environmental Safety, Volume 74, Issue 5, 2011
- Rodríguez, A. et al. (2008). Ozone-Based Technologies in Water and Wastewater Treatment. In: Barceló, D., Petrovic, M. (eds) Emerging Contaminants from Industrial and Municipal Waste. The Handbook of Environmental Chemistry, vol 5 / 5S / 5S/2. Springer, Berlin, Heidelberg.
- Sourav Mondal, Mihir Kumar Purkait, Sirshendu De (2018). Advances in Dye Removal Technologies.
- MohanKumar, S. M. J., Balasubramanian, P., Subramanian, M., & MohanKumar, P. S. (2018). Chronic estradiol exposure – harmful effects on behavior, cardiovascular and reproductive functions.
- James D. Finnigan, Carl Young, Darren J. Cook, Simon J. Charnock, Gary W. Black, Chapter Nine - Cytochromes P450 (P450s): A review of the class system with a focus on prokaryotic P450s
- Ulrich M. Zanger, Matthias Schwab, Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation, Pharmacology & Therapeutics
- Smith LB, Tyagi R, Kasai S, Scott JG. CYP-mediated permethrin resistance in Aedes aegypti and evidence for trans-regulation.
- Mao W, Schuler MA, Berenbaum MR. CYP9Q-mediated detoxification of acaricides in the honey bee (Apis mellifera).
- Han H, Yu Q, Beffa R, González S, Maiwald F, Wang J, Powles SB. Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.
- SD (2018) Birth of a photosynthetic chassis: a MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii
