File:TUM13 General principle of accumulation of persistent chemicals along the food chain.jpg
Figure 1: General principle of accumulation of persistent chemicals along the food chain.

Bioaccumulation is a difficult concept to define. The U.S. Environmental Protection Agency, 2010 defines bioaccumulation as a “general term describing a process by which chemicals are taken up by an organism either directly from exposure to a contaminated medium or by consumption of food containing the chemical”. The majority of definitions is aimed at the build up of persistent chemicals such as DDT, PBC or dioxins along the food chain (see figure 1), where the pollutants accumulate in predators until they become toxic. This can lead to the local and global extinction of species and health problems for humans. As most of these persistent chemicals are not biodegradable a different approach is necessary.

Figure 2: Principle of our bioaccumulation system.

Using our transgenic moos (Physcomitrella patens ) as a chassis, we can produce effectors for specific binding of pollutants. For the sustained removal of the target pollutant the effectors must be membrane-anchored, in contrast to the cytoplasmic or secretory localization of biodegrading effectors. The membrane association ensures the fixation of the pollutant to the membrane after successful binding. Internalisation of the effectors either results in metabolism or accumulation of the pollutant in the transgenic plant cell. In case of accumulation, controlled disposal of the plants in the filter would be necessary.

There is a broad range of natural as well as engineered binding proteins available. Natural binding proteins act as a model and initiator in design of new artificial binding proteins combining research in different fields of biotechnology. Lipocalins, natural binding proteins, as base and scaffold for the design of anticalins confirm the popularity of bioaccumulated proteins in red biotechnology [Schlehuber et al., 2005]. The role of Fibrillin in the abscisic acid-mediated photoprotection shows an example of functioned bioaccumulation in a plant [Yang et al.,2007]. The most commonly known binding proteins are antibodies which defend mammals against pathogens and toxins. Besides these natural binding proteins and anticalins there are more and more designed binding proteins such as Affibodies derived from the z-domain of the antibody-binding protein A (Ref) and DARPins that are based on an ankyrin scaffold. One research topic of the chair of analytical chemistry of the TU Munich is the depletion of algal toxin contaminated water by using selective biofilters based on plantibodies. Plantibodies are plant- produced and derived antibodies which can construct antibody-mediated pathogen resistance as well as change the plant phenotype by immunomodulation [Stoger et al.,2002].

For our project we exemplarily choose three different binding proteins which each exploit different mechanisms to bind Proteins:

  • The anticalin fluA as an example for a binding protein.
  • The enzyme Gluthathione-S-Transferase as an example where the formation of covalent bonds between the target pollutant and another molecule (gluthatione) is catalized.
  • The enzyme Protein Phosphatase 1 as example for a target of a binding protein. Here we exploit the inhibitory binding of the pollutant to the enzyme.

To sketch the theoretical binding to a membrane anchored protein in our video, we created a short animation of the process using the molecularMaya software:

Bioaccumulation via binding proteins

Problem Example: Diclofenac

Are pharmaceuticals a blessing or a curse? Respective to the ecological consequences of diclofenac this task must be valued negative. Diclofenac which is a proven, nonsteroidal anti-imflammatory drug (NSAID) is used mostly in human medicine to treat a variety of acute and chronic pain and inflammatory conditions via inhibition of prostaglandin synthesis by inhibiting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)[Gan,2010]. Its necessity and importance in medicine research confirm e.g. the development of diclofenac patches for topical treatment of acute impact injuries [Predel et al.,2004] and the main studies about safety and efficiency of the analgesic, anti-inflammatory, and antipyretic properties of diclofenac [Morgan et al.,2001][Chan et al.,2004][Pavelka,2012]. The possible resulting vicious circle (see Figure 1) shows the poisoning tragedy in 2005 which led to mass mortality and decline of vultures species especially Gyps bengalensis, Gyps indicus and Gyps tenuirostris across the Indian sub-continent. Based on the consumation of diclofenac by animals, vultures have an abnormally high intake in this drug by eating the cadavers of contaminated animals.

Current solutions

The fate of degradation products with their toxicity character and the development of antibiotic resistances plays a major role of the research on the effect of diclofenac in the aquatic environment. Because of the lack of analytical standards and the complex matrix of organic molecules in environmental samples, the degradation products have not yet been identified regarding molecular structure or concentration in aquatic fauna and flora.

The degree of the problem becomes visible in HPLC analysis, which shows in addition to the fast degradation of diclofenac over the irradiation time, a phytotoxicity increase of the increasing degradation product concentrations and a weak inhibition of cell growth.

To reduce the existence of PhACs such as diclofenac, one possibility is the usage of sewage treatment plants (STPs). Their removal efficiency depends on the sludge retention time and on the characteristics of the pharmaceutical molecule. Other strategies are the adding of an additional stage to the STP. Another approach is treated in the publications [Westerhoff et al. (2005)][Huber et al., (2005)][Ternes et al., (2003)] which confirm a high removal efficiency up to 95% for these additional stages. Currently developments are the nano-filtration, ozonation and power activated carbon. The importance of knowing the transformation products before applying a new technology is again demonstrated by the fact that ozonation reduces the parental substance diclofenac but cannot mineralize it.

To sum it up, biodegradation of diclofenac introduces several new problems as degradation products are toxic and pose new harms to the environment.

Bioaccumulation instead of Biodegradation

Figure 3: Structure of fluA(blue) with its ligand fluorescein(green) [source:PBD]

As biodegradation does not conclusively solve the problem of diclofenac in the aquatic environment, we propose the utilisation of antibodies for accumulation of diclofenac, which would solve the problem of toxic degradation products.

Although an antibody against diclofenac has been developed [Deng et al. (2003)], in vivo verification of successful binding would not be straightforward. Therefore we aimed for a first proof of principle and decided to investigate another substance that is targetable by antibodies for which verification is feasible. One attractive option is the well-known fluorophore fluorescein which was used by the team Freiburg 2008. Fluorescein fluoresces until bound by the binding protein fluA, which makes characterization tractable. The covalent binding of fluA and flourescein is shown in Figure 3.

We submitted an improved version of the Freiburg Biobrick for fluA (see Table 1) and created a transgenic moss plants (PF-15) with the fluorescein binding anticalin fluA on the extracellular part of the receptor.

Producing engineered binding proteins for medical purpose in the red biotechnology is one of the main research fields in which anticalins play a major role. Lipocalins such as bilin-binding-protein BBP (from Pieris brassicae) can be used for generating molecular pockets with a diversity of shapes and for creating a stable receptor protein for a ligand of choice, so that development of binding proteins against nearly chemical structures with comparable size is possible. Hence it should be possible to target a great variety of pollutants by already available binding proteins.

Table 1: Variants of the fluorescein binding Anticalin FluA
Proteinvariant KD of FluA to fluorescein Literature reference BioBrick
FluA 152 nM [Beste et al., 1999] <partinfo>BBa_K157004</partinfo>
FluA (R95K) 64 nM [Vopel et al., 2005] not available as BioBrick
FluA (R95K, A45I, S114T) 2 nM [Vopel et al., 2005] <partinfo>BBa_K1159002</partinfo>


Problem: Dichlorodiphenyltrichloroethane (DDT)

Since the entry into force of the Stockholm Convention in 2004 the insecticide Dichlordiphenyltrichlorethan (DDT) is only allowed for the abatement of disease-carrying insects such as the malaria carrying Anopheles dirus mosquito [La-Aied Prapanthadara et al.,1996]. This measure was the consequence to the devastating consequences for the ecosystem by the widespread use of DDT, which continuously grew due to it's strong insecticidal properties. Despite the regulation in 2004, DDT still poses an environmental threat today due to its stability and capacity to accumulate in tissue [Turusov et al.,2002].

Current Solution

The simultaneous degradation of mixed insecticides like DDT can be achieved by mixed insecticide enriched isolated cultures in media and soil such as the mixed fungal population (white rot fungus Phanerochaete chrysosporium). The degradation efficiency of DDT can be enhanced by using carbon (mannitol) and nitrogen (sodium nitrate) in the liquid media. Based on the disadvantages which bring along the diverse strategies like chemical treatment, incineration, and landfills the development of the so called soil microflora method leads to a positive effect concerning to the detoxification of pesticides. The poor fungal growth in the media and concomitant biodegradation of insecticides is caused by toxicity exerted by high concentrations of the substrate which explains the low degradation at high concentrations.

Caltech 2011 Solution: Covalent binding to Gluthathion via Gluthathione-S-Transferase

Figure 4: Conjugation of GSH and a xenobiotic

In context to prove the bioaccumulation and to develop an effective methode of removing DDT, we envision the usage of the cytoplasmatic GST 1-1, also known as DDT Dehyrochlorinase. Along with the creation of transgenic Physcomitrella patens plants (PF-13) we used the biobrick from the Caltech iGEM team 2011. This team engineered bacteria which can degrade endocrine-disrupting chemicals such as DDT, synthetic estrogen in bodies of water to less toxic forms. Glutathion S-transferases (GSTs), an eukaryotic and prokaryotic phase II metabolic isozymes-family, catalyze the conjugation of reduced form of gluthatione (GSH, nucleophil) and xenobiotics (electrophil) to gluthatione-S-Conjugate via nucleophilic attack (see Figure 4).

Figure 5: Equilibrium reaction of gluthation

The consequence is an increased solubility of the conjugates which leads to the removal of xenobiotics in form of conjugates via vacuole enclosure. The action of the specific transporters and the steady supply of GSH in the equilibrium reaction (see Figure 5) are the limited factors of the detoxification reaction. Along with the detoxification and cell signaling function GST’s act as transport proteins, which gave GST the previous name ligandin. Table 2 shows the 3 different superfamilies with their characteristics.

File:TUM13 Structure3D of GST.gif
Figure 6: Molecular structure of Gluthation S-Transferase
Table 2: Superfamilies of GST with their characteristics
Superfamily of GTS Classes based upon their structure Sequence homology [%]
Cytosolic proteins alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega >40
Mitochondrial proteins kappa <25
microsomal (MAPEG= membrane-associated proteins in eicosanoid and glutathione metabolism) proteins subgroups I-IV <25
Figure 7: Reaction of monochlorobimane with GSH

Generally the three superfamilies differ mostly in structure and sequence as only the cytosolic and the mitochondrial superfamily have a thiorexin like domain in which the glutathione binding site (G-site) is located [Oakley A.,2011]. The helix alpha 2 is the most variable secondary structure. Y-GST is the subgroup which activates glutathione via using tyrosine residues. S/C-GST uses serine/cysteine residues. GST binds the substrate at the hydrophobic H-site of the enzyme and GSH at the hydrophilic G-site which together form the active site of the enzyme (see Figure 6). In research techniques GST is used as so called GST-tags for separation, elucidation of direct protein-protein interaction and purification of the GST-fusion protein mostly by pull-down assay. So targeting GST with molecule therapeutics represents GTS as an attractive target for drug discovery [McIlwain et al.,2006].

A mammalian variant of GST, GSTP, plays a major role in cancer- development and potential drug/chemotherapeutic resistance in a majority of tumor cell lines: The inhibition of the pro-apoptotic pathway (JNK pathway) and the overexpression of GSTP in tumor cells lead to escape of apoptosis of the tumor cells mediated by non GSTP- substance-drugs [Hayes et al.,1995] [Josephy,2010] [Hayes et al.,2000] [Fraser et al.,2003]. To avoid the time- and labor-intensive method HPLC following derivatization with 2-nitrobenzoic acid we used the common sensitive technique with monochlorobimane to measure GSH as a proof of principle. The adding of monochlorobimane to the culture medium leads to the conjugation of GSH to monochlorobimane catalyzed by DDT Dehyrochlorinase (see Figure 7). The GSH-monochlorobimane conjugate can be measured fluorometrically [Kamencic et al.,2000].

Protein Phosphotase 1 – A molecular mop for Microcystin

The Problem: Algal Blooms

Figure 8: Eutrophication

Which phenomenon implicates the common name “Florida red tide”? This term is used in annually along Florida waters where species known as Karenia brevis leads to a red colored alga bloom along the Florida coast. Algal blooms are a worldwide environmental problem! This becomes evident with the appointment of 10.000 people for cleaning up the algal bloom in Beijing for the Olympic discipline “sailing regatta” in 2008. Depending on the cause, bacteria algal can bloom at concentrations of hundreds to thousands of cells per milliliter. An officially recognized threshold level isn’t public. Possible diverse factors which are the reason for the high affecting of bloom formation are listed followed:

  • Light intensity: cyanobacteria which form surface blooms have a higher tolerance concerning high light intensities. This leads to lower growth rate than other phytoplankton organisms.
  • Gas vacuoles: Gas vesicles give the cyanobacteria a lower density than water.
  • Growth rate: Slow growth rates require long water retention times to enable a bloom.
  • Phosphorous and nitrogen: A low ratio of these substance may favour a development of cyanobacteria blooms.
  • Temperature: maximum growth rates are attained by the most cyanobacteria above 25°C. Thus much bloom formation is during the summer.

A possible cascade which is caused by the release of nutrients in the aquatic ecosystems is shown in Figure 8. Another result confirms the spread and the persistence of cholera through algal blooms [Epstein PR.,1993]. Currently the so called harmful algal blooms are an additive for the detection of bacteria spread and their caused health effects to fauna and flora [Ferrante et al.,2010] [Barlaan EA et al.,2007]. The reduction of the uptake of toxic methylmercury in freshwater food webs by algal blooms confirms a positive reverse of algal blooms [Pickhardt PC. et al.,2002].

Current Solution to combat Algal blooms

It is well known that cyanobacteria produce most Toxins such as microcystin under species-specific conditions which are most favorable for their growth. As a result most studies concerning this topic try to tackle the problem from this point of view. For the investigated species, they found out that the toxin production increased under light optimum condition of the relative bacteria species and on low and high pH. The usage of zinc was necessary for the increase of growth and toxin production. Dependent on nitrogen fixing respectively non-nitrogen fixing species the nitrogen and phosphorous rate in media was unnecessary respectively necessary concerning to the toxin production. Evidences of the role of plasmid, multi-enzyme complexes and peptide synthetase gene involvement in toxin production were also confirmed: e.g. knockout experiments showed that peptide synthetase genes are responsible for microcysteine production.

Major problems are still the lack of knowledge regarding to occurrence and toxicity of cyanobacterial products like the diversity of LPS structures which cause allergic as well as toxic human health problems, the extremely weak scientific basis and poorly developed procedures for valuation on health effects. The TDI (tolerable daily intake) is one of the key features on which the toxicology studies are aimed. Based on analytic and sampling problems or inadequate scientific data safe practice guidelines can be assisted for the reducing of exposure.

The human exposure to the toxins is a major point to combat the human health risk. The WHO guideline values base upon a number of assumptions which change locally or nationally according to the present situation. To fill the lack of knowledge of parental variants the HPLC method is commonly used also for the determination of references. Current treatment processes used at water surface plants are coagulation, sand filtration, and clarification.

The negative site of these methods is the ineffective removal and destruction of cyanotoxins. To combat this problem treatment systems like carbon- and membrane filtration were developed. External and internal accumulation of toxins is another research topic since the inhibition of Mustard seedling development by of microcystein-LR in aquatic root solution.

Dundee 2013 Solution to combat Algal blooms

Figure 9: interaction of PP1 and microcystin [source: PDB]

Due to the deleterious results from the toxin release of algal blooms many risk assesments were have been developed [Schaeffer et al., 1999]. The effects of microcystin for the aquatic and terrestrial ecosystem have been proven to include the modulation of proliferation, apoptosis and proliferation of spermatogenic cells in vivo [Zhou et al.,2013], which makes it an attractive target for our project.

For the bioaccumulation of microcystin, it is possible to use the enzyme PP1. Mycrocystin can inhibit the function of PP1 by binding to it, this interaction of microcystin, in form of a heptapeptide structure, with three distinct regions of the PP1c is depicted in Figure 9. Hence PP1 can act as natural binding partner for mycrocystin.

As the Dundee iGEM team 2013 Team Dundee was already working on this subject we contacted them and they kindly provided us with the gene that encodes PP1. After converting the human PP1, to RFC 25 and constructing some expression plasmids we produced transgenic phytomitrella patens (PF-14) with PP1 and a biobrick of PP1 receptor.


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