HebrewU HujiGEM 2018

Hebrew

Project
- Description
- Model
- Results
- Parts
- Moolti
Lab
- Yeast Design
- Plant Design
- Notebook
- Inter-lab
- Safety
- Protocols
Human Practices
Team
Judging

HebrewU HujiGEM 2018

Hebrew

Project
- Description
- Model
- Results
- Parts
- Moolti
Lab
- Yeast Design
- Plant Design
- Notebook
- Inter-lab
- Safety
- Protocols
Human Practices
Team
Judging

The Problem

For over a century , dioxins – and specifically chlorinated dioxins – have been notorious as some of the most toxic and persistent environmental pollutants. Though some dioxins occur naturally, 2,3,7,8-tetrachlorodibenzodioxin (TCDD), the most toxic chemical of this family, is entirely synthetic. These compounds are extremely stable and, due to their synthetic nature, many are not easily broken down by living organisms. Dioxins remain in our ecosystems for decades, transferring from soil to plant, to animal, and back to the soil again in an unending cycle.

The toxicity of TCDD is such that varying doses of the toxin had caused cancer in all animals tested. TCDD’s effect is widespread, reaching almost all of the internal organs, depending on the type of animal that is exposed. Birth defects as well as severe developmental issues have been observed in humans as well as other animals. TCDD is more toxic than cyanide, ricin and even plutonium.

How is TCDD created?

TCDD is not, and has never been produced commercially except as a pure chemical for scientific research. It is, however, a byproduct of chlorophenols or chlorophenoxy acids which are produced as herbicides and fungicides. It may also be formed along with other polychlorinated dibenzodioxins (PCDD’s) and dibenzofuranes (PCDF’s) during incineration, especially if certain metal catalysts such as copper are present. Generally, small amounts of PCDD/Fs are formed whenever organic materials such as oxygen and chlorine are available at suitable temperatures. As a result of this, when organic material is burned in less-than-optimal conditions- open or building fires, domestic fireplaces, and poorly operated and designed solid waste incinerators- these chemicals are synthesized at the highest high rates. Historically, municipal and medical waste incineration was a central source of PCDD/Fs.

Other sources of PCDD/F include:
  ·Metal smelting and refining.
  ·Chlorine bleaching of pulp and paper - historically important source of PCDD/Fs to waterways.
  ·Synthesis side products of several chemicals, especially PCBs, chlorophenols, chlorophenoxy acid herbicides and hexachlorophene.
  ·Uncontrolled combustion, particularly open burning of waste ("backyard barrel burning"), accidental fires, wildfires.
  ·Engines using leaded fuel, which contained the additives 1,2-Dichloroethane and 1,2-Dibromoethane, (a practice no longer used).

Mode of action

TCDD and dioxin-like compounds act via a specific receptor present in all mammalian cells: the aryl hydrocarbon (AH) receptor. This receptor is a transcription factor which is involved in expression of genes; in fact, it has been shown that high doses of TCDD affects expression of several hundred genes in rats, increase some while decreasing others. Research shows a particularly strong effect on transcription of enzymes activating the breakdown of foreign and toxic compounds.

Polycyclic hydrocarbons also activate the AH receptor, but less than TCDD and only temporarily. Even many natural compounds (present in certain vegetables, for instance) cause some activation of the AH receptor. This phenomenon can be viewed as adaptive and beneficial, because it protects the organism from toxic and carcinogenic substances. Despite this, excessive and persistent stimulation of AH receptor, leads to a number of adverse effects.

While the mutagenic and genotoxic effects of TCDD are sometimes disputed, it has been confirmed that it does foster the development of cancer. Its main action in causing cancer is promoting the carcinogenicity initiated by other compounds. Very high doses may, in addition, cause cancer indirectly; one of the proposed mechanisms is oxidative stress and the subsequent oxygen damage to DNA. There are other explanations such as endocrine disruption or altered signal transduction.

We devoted ourselves to this issue because to date, the existing solutions are astoundingly impractical and inefficient. The solutions being employed address only local-scale pollution, and are not sustainable.

The biggest TCDD remediation project today, is happening in Vietnam. During the American-Vietnam war in the 1960’s, tens of thousands of kilometers of land were heavily sprayed with an herbicide called Agent Orange. But TCDD was an unintended byproduct of Agent Orange production; thus, the military had unknowingly contaminated all of that land with this toxin. Today, over 50 years after the events took place, the United States is spending hundreds of millions of dollars to reclaim this land.

Today, soils in Vietnam are being decontaminated by transporting only the most heavily contaminated soils to a castle-sized, makeshift “oven”. Once there, the soil is incinerated 350°C for months. Although this method successfully rids soils of their dioxin contaminants it is impractical for a number of reasons.

1. Only so much soil can be gathered for purification.
2. The process is detrimental to the soil, damaging both microbial communities and soil structure.
3. This method is unsustainable, and cannot be used for large scale pollution over large areas.

Additionally, is important to note that dioxin pollution is found in small amounts nearly everywhere in the world. Soil-burning would be far too inefficient and expensive to deal with these types of contaminations.

Our Solution

The biological solution we have designed is a novel enzymatic pathway capable of breaking down TCDD into harmless metabolites. We have identified a group of enzymes derived from different organisms that, when combined, completely degrade TCDD. We incorporated these enzymes into a single transgenic plant that can breakdown TCDD and dioxins, as well as detoxify chlorinated pollutants.

We chose plants as the host for this pathway for several reasons:

1. Plants are easy to track and control. Through various genetic and molecular methods, we can sterilize the plants to ensure our GMO’s don’t invade the ecosystem.

-By contrast, microbes require more intricate and complicated control mechanisms that hinder efficiency and flexibility regarding the implementation of our research.

2. Plants possess specific attributes that are critical to our project’s success.

-Plant root systems are efficient at accumulating dioxins from soil and water.
-Plants naturally contain useful enzymes, that aid in the breakdown of TCDD.

3. Utilizing plants, which sit at the bottom of the food chain, we can keep animals in polluted areas safe, and decrease the bioaccumulation of TCDD.

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Our team has created an exploratory model of a metabolic pathway for the enzymatic degradation of TCDD. This model served as the foundation of our project design and the backbone for our lab work. We completed in depth research on enzymes families relevant to TCDD degradation, narrowing down contenders to the enzymes we would eventually use in the lab. While building this model, we utilized a multitude of tools including, but not limited to biochemical analysis, phylogenetic comparison and pathway mapping. Through these tools, we compiled a comprehensive list of enzymes, is strongly rooted in prior academic studies.

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The final pathway we tested in the lab, consists of dehalogenase, dioxygenase and hydrolase enzymes. Additionally, Cyp 450 and Lignin Peroxidase enzymes were researched, enzymes have been privously found to breakdown TCDD. Despite that neither of these enzymes catalyze a specific step in the final engineered pathway, both are excellent candidates for TCDD Degradation.

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Cytochrome P450

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Collectively, the involvement of P450s has been proven in the metabolism of aliphatic, alicyclic, and aromatic molecules in reactions resulting in hydroxylation, epoxidation, dealkylation, sulfoxydation, deamination, desulphuration, dehalogenation, and N-oxide reduction. The majority of P450s catalyze the reactions after interacting with one or more protein components which transfer electrons from NADH or NADPH to the P450s, while some of them do not require any additional protein components to achieve the reductive activation of oxygen. While bacterial CYPs are water-soluble, mammalian CYPs are bound to either ER or mitochondrial membranes. ^1,2

Mammalian CYP450:

Though human CYP's showed the ability to breakdown PCD's, it appeared that they were unable to utilize 2,3,7,8 TCDD as substrate. By contrast, CYP450 deriving from canines have showed some ability to catalyze hydroxylation of 2,3,7,8-TCDD. In 2003, an article published by Kyoto university, described a single amino acid mutation [F240A] which allowed a rat CYP enzyme to catalyze TCDD dechlorination in E. coli.³

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In vivo studies suggest that the CYP-dependent metabolism includes multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and opening of the dioxin ring. All of these reactions appear to be detoxication reactions of PCDDs.

Aromatic hydroxylation reactions of arenes, 7, are catalyzed by P450. The initial product is a reactive arene epoxide, 8, which then undergoes an interesting rearrangement (NIH shift) to yield a reactive keto 2,4-diene intermediate, 11. This reactive intermediate rapidly undergoes a tautomeric shift of a proton to restore aromaticity.

Plant CYP450:

Cytochrome P450 monooxygenases (P450s) represent the largest enzyme family of the plant metabolism. Plants typically devote about 1% of the protein-coding genes for the P450s to execute primary metabolism and also to perform species-specific specialized functions including metabolism of the triterpenes. The members of the subfamilies CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A are generally conserved across the plant kingdom to take part in plant primary metabolism related to the biosynthesis of essential sterols and steroid hormones. However, the members of the subfamilies CYP51H, CYP71A,D, CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y are required for the metabolism and might perform species-specific functions including chemical defence toward specialized pathogens.⁴

Fungal CYP450:

Cytochrome P450 monooxygenases of fungi are involved in many essential cellular processes and play diverse roles. The enzymes catalyze the conversion of hydrophobic intermediates of primary and secondary metabolic pathways, detoxify natural and environmental pollutants and allow fungi to grow under different conditions. Fungal genome sequencing projects have enabled the annotation of several thousand novel cytochromes P450, many of which constitute new families. Previous reports demonstrated that the white-rot fungus P. chrysosporium has been shown to possess biodegradative capabilities 2,3,7,8-TCDD. The degradation of more highly chlorinated DDs by P. chrysosporium has also been demonstrated. Based on the fact that P. chrysosporium genome contains 148 CYP genes, it is possible to assume that some P. chrysosporium CYPs are involved in the metabolism of PCDDs. Unfotunately, The specific 2,3,7,8-TCDD-metabolizing enzyme has not yet discovered in P. chrysosporium.^2,5

Prokaryotic CYP450:

Bacterial CYP102A1 is a water-soluble fusion protein between CYP and NADPH-P450 reductase containing FAD and FMN. Interestingly, CYP and the reductase domains of CYP102A1 are significantly homologous with animal CYP and its reductase, suggesting it maybe the eukaryotic origin of CYP102A1 (Omura 2010). These cytochromes are involved in the synthesis of secondary metabolites, such as antibiotics, in the utilization of hydrophobic low molecular substrates, such as alkanes and aromatic carbohydrates. These enzymes also have monooxygenase activity towards different organic low molecular substrates. Cytochromes C450 play an important role in the degradation of environmental toxins and mutagens. The hydroxylation activities of these enzymes towards PCDDs are much lower than those of mammalian CYPs.¹

References:

1. <a href="https://link.springer.com/article/10.1007/s00253-010-2765-2">"Enzyme systems for biodegradation of polychlorinated dibenzo-p-dioxins"; Toshiyuki Sakaki et. al, Applied Microbiology and Biotechnology September 2010, Volume 88, Issue 1, pp 23–30. </a>
2. <a href="https://www.ncbi.nlm.nih.gov/pubmed/20619366">"Cytochrome P450 enzymes in the fungal kingdom"; Crešnar B et. al, Biochim Biophys Acta. 2011 Jan;1814(1):29-35. </a>
3. <a href="https://link.springer.com/article/10.1007/s00253-005-0286-1">"Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant"; Raku Shinkyo, Applied Microbiology and Biotechnology, September 2006, Volume 72, Issue 3, pp 584–590. </a>
4. <a href="https://www.frontiersin.org/articles/10.3389/fpls.2017.01886/full">"Triterpene Structural Diversification by Plant Cytochrome P450 Enzymes";Sumit Ghosh, Front. Plant Sci., 09 November 2017 </a>
5. <a href="https://www.sciencedirect.com/science/article/pii/S1087184597910215">"Cytochrome P450 Enzyme Systems in Fung"; Hans J.M et. al, Fungal Genetics and Biology Volume 23, Issue 1, February 1998, Pages 1-17 </a>

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Dioxygenase

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Many of these reactions have little or no precedent in organic chemistry. Di-oxygenase and mono-oxygenase enzymes that are able to activate dioxygen are divided into oxidases, which use oxygen as an oxidant, and reduce dioxygen to hydrogen peroxide or water; and oxygenases, which incorporate oxygen atoms from dioxygen into the product(s).Dioxygenases incorporate both atoms of oxygen into the product(s).

                       
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Oxidization of Dibenzofuran, a dioxin like molecule we used in the lab1 ¹.

Most dioxygenase enzymes require a metal cofactor, which is most often iron(II or III). Dioxygenases primarily oxidize aromatic compounds and, therefore, have applications in environmental remediation. Dioxygenases serve as part of nature’s strategy for degrading aromatic molecules in the Environment. They are often found in the soil bacteria as well as almost all higher plants. Dioxygenase enzymes are used for a multitude of separate steps in the pathway described.

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Proposed initial dioxygenolytic attack on the tested CDFs and CDDs by DFDO and CARDO. Panel 1, 2-CDF, 5′-chloro-2,2′,3-THB (reaction A) and 5-chloro-2,2′,3-THB (reaction B); panel 2, 2,8-DCDF, 5,5′-dichloro-2,2′,3-THB (reaction A); panel 3, 2-CDD, 5′-chloro-2,2′,3-THDE (reaction A), 4′-chloro-2,2′,3-THDE (reaction A′), 5-chloro-2,2′,3-THDE (reaction B), and 4-chloro-2,2′,3-THDE (reaction B′); panel 4, 2,3-DCDD, 4′,5′-dichloro-2,2′,3-THDE (reaction A) and 4,5-dichloro-2,2′,3-THDE (reaction B); panel 5, 2,7-DCDD, 4,5′-dichloro-2,2′,3-THDE (reaction A) and 4′,5-dichloro-2,2′,3-THDE (reaction B); panel 6, 1,2,3-TCDD, 4′,5′,6′-trichloro-2,2′,3-THDE (reaction A), 3′,4′,5′-trichloro-2,2′,3-THDE (reaction A′), and 4,5,6-trichloro-2,2′,3-THDE (reaction B). Possible sites attacked by angular dioxygenases are indicated by A or A′ on the nonsubstituted rings and by B or B′ on the halogen-substituted rings, with the exception of those in panels 2 and 5. ND, not detected. ²

Numerous studies have been done on a number of Dioxygenase enzymes, and their ability to degrade dioxins:

References

1.<a href="https://www.semanticscholar.org/paper/Dibenzofuran-4,4a-dioxygenase-from-Sphingomonas-sp.-B%C3%BCnz-Cook/3d6600bf8164f0d6570ca9d7208f865607cdd631">"Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system"; Patricia V. Bünz et. al, Journal of bacteriology 175 20 (1993): 6467-75.7313–7320 </a>
2.<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC93062/"> "Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Two Types of Bacteria Having Angular Dioxygenases with Different Features";Hiroshi Habec et. al, Appl Environ Microbiol. 2001 Aug; 67(8): 3610–3617. </a>

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Peroxidase

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White rot fungi, the fungi responsible for the biodegradation of lignin in wood, are remarkable in their ability to degrade a wide variety of environmental pollutants. Insoluble, generally very recalcitrant chemicals are mineralized by the fungi. The fungi also mineralized (oxidized to CO2) some chemicals that are already highly oxidized. In general it is thought that this biodegradative ability is related to the ability of these fungi to degrade lignin. This ability, which is unique to white rot fungi, is thought to be dependent on a family of peroxidases secreted by the fungi; however, a purely oxidative, peroxidase- based system cannot be used to completely degrade lignin. Lignin is highly oxidized so it is difficult to oxidize further. Lignin is a complex heteropolymer with no stereochemical regularity, due at least in part to the free radical mechanism of synthesis. Lignin biodegradation must therefore involve a nonspecific and non-stereoselective mechanism.

The lignin peroxidases are somewhat unique in that they have higher oxidation potentials (- -1.35 V) than do most peroxidases ( -0.8 V). In this way these enzymes have a somewhat greater range of chemicals that they can oxidize. Due to this none specific, and highly effective oxidation abilities, Lignin peroxidase is an excellent candidate for bioremediation- not only for dioxins, but a wide range of aromatic pollutants.¹

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The C-O--C bond cleavage occurs via the nucleophilic attack of water on a cation rather than via the scavenging of a radical by molecular 02. The first step in this proposed mechanism is the one-electron oxidation of I by the oxidized enzyme intermediate LiP compound I, resulting in the formation of the aryl cation radical A. The LiP oxidation of a variety of nonphenolic aromatics to their corresponding aryl cation radicals has been well established. Indeed, the LiP oxidation of the related nonchlorinated compound dibenzop-dioxin to a cation radical has been reported. However, DCDD is considerably less soluble than dibenzo-p-dioxin. Furthermore, chloride is a much better leaving group than hydrogen; therefore, the aryl cation radical A is probably short-lived. For these reasons, the cation radical A was not detected during the oxidation of DCDD by LiP.

Attack of H20 at the cation would result in the loss of chloride and the formation of the carbon-centered radical intermediate B. One-electron oxidation of B by LiP or MnP would result in the formation of the cation intermediate C. Attack of H20 on intermediate C would lead to the first C--O-C bond cleavage and the formation of the quinone intermediate D. Subsequent oxidation of the phenolic function of D would generate the phenoxy radical E, which is in resonance with the carbon-centered radical E'. Oxidation of E' by either LiP or MnP would yield the cation F. Finally, attack of H20 on the cation F would result in the cleavage of the second C-O-C bond and generation of 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). In the metabolic pathway, both chlorine atoms are removed from the aromatic rings as chloride before ring cleavage takes place. This is advantageous because the possible formation of toxic chlorinated aliphatics is avoided. Indeed, peroxidasecatalyzed oxidative dechlorination followed by reduction of the quinone products results in the introduction of phenolic groups, which facilitate subsequent ring opening. Similar peroxidase-catalyzed oxidative dechlorinations of polychlorinated phenols have been reported.
We propose that a similar metabolic pathway is probably involved in the fungal degradation of TCDD and have initiated studies to examine its metabolism.²

References:

1. <a href="http://science.sciencemag.org/content/228/4706/1434">"Oxidation of persistent environmental pollutants by a white rot fungus"; JA Bumpus et. al, Science 21 Jun 1985: Vol. 228, Issue 4706, pp. 1434-1436.</a>
2. <a href="https://jb.asm.org/content/jb/174/7/2131.full.pdf">"Degradation of 2,7-Dichlorodibenzo-p-Dioxin by the Lignin Degrading Basidiomycete Phanerochaete chrysosporium "; K Valli et. al, Journal of Bacteriology, Apr. 1992, p. 2131-2137</a>

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Team:HebrewU/Model

Contents

Further Reading:

References:

Mammalian CYP450:

Further Reading:

Plant CYP450:

Fungal CYP450:

Prokaryotic CYP450:

References:

Further Reading

References

Further Reading:

References:

Further Reading:

References: