Team:Copenhagen/Project

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Table of Contents

Sponsors

CyperMan to the

Rescue

CyperMan - Kills fungus

We hope to be able to develop bacteria that can kill fungus.
We will introduce a biological system to ''E.coli'' utilizing the plant cytochrome p450 79’s ability to produce small molecules called oximes which are toxic to fungus, as they inhibit the ability of the mitochondrial peroxidases to breakdown potentially harmful hydrogen peroxides. The oximes are produced from aminoacids - phenylalanine, tyrosine and tryptophan.

CyperMan - Saves the Fish

We hope to find and possibly optimize a biological system which can be introduced in bacteria and lead to the removal of PPCP (pharmaceutical and personal care products) pollutants in water. We intend to transform E.coli bacteria with different combinations of the several membrane bound human cytochrome p450 and examine our different cytochrome p450 transformed E.coli bacteria and their ability to impede estrogenic activity water.

Cytochrome p450 CYP 1 & 2 - the source of CyperMans water cleansing power

Cytochromes p450 is one of the largest gene superfamilies coding for enzymes present in the genomes in all biological kingdoms [1]. The enzymatic activities of these proteins are extremely diverse with activity in biotransformation of drugs, bioconversion of xenobiotics, biosynthesis of compounds as steroids, fatty acids, eicosanoids, fat soluble vitamins and bile acids. Furthermore, cytochrome p450’s are involved in the conversion of alkanes, terpenes and aromatic compounds as well as degradation of herbecides and insecticides [1].

We will exploit this activity of cytochromes p450 in the bioconversion of xenobiotics to neutralize damaging medical residues in waste water.

Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name [2]. Despite the label as cytochromes, these proteins are not involved in electron transfers, but act as monooxygenases in a wide range of reactions such as epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation [3].

We will focus on the hydroxylating property of the cytochromes, which is also the defining reaction for these enzymes. The reductive activation of molecular oxygen reduces one of the oxygen atoms to a molecule of water, as the other is inserted into the substrate [2].

Fig 1

RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O

A key donor of electrons for the reduction of molecular oxygen is the NADPH dependent cytochrome p450 reductase (CPR). This protein shuttles electrons from NADPH through the FAD and FMN- coenzymes into the iron of the prosthetic heme group of cytochrome p450 [4].

A characterization of the cytochromes p450 is the main objective with this project. We will exploit the wide specificity of these cytochromes to target many different damaging agents continuously by hydroxylation, which can reduce the damaging activity of e.g. estrogen.

**Figure 1**The P450 catalytic cycle in hydroxylation

Impact factors:

Open source BioBricks:

Our project has the aim to characterize different subfamilies of Cytochrome p450 as a BioBrick and thus add it to the partsregistry of BioBricks, a continuously growing collection of genetic parts that can be mixed and matched to build synthetic biology devices and systems (partsregistry.org). Hence our project contributes to the expansion of the partsregistry, benefitting all researchers with interest in these particular proteins. The partsregistry is founded on an open source philosophy, a philosophy we see as a great advantage for development and improvement of the number BioBricks already existing. The addition of different cytochrome p450 to this catalogue will perhaps encourage other groups to look at other aspects of the many functions of p450's and thus contribute to the formation of new biological systems – further expanding the field of synthetic biology.

Ecological and economical prospects:
Another future aim with our project is to improve wastewater treatments and thus contribute to a cleaner and uncontaminated drinking water for everyone. With this scientific project, we will move towards this realization, but more research is needed. Our belief is that further research on the use of cytochrome p450 will give a valid and an inexpensive product for use in wastewater treatment plants, thus benefitting the environment and our planet.

References

[1] F. Hannemann, A. Bichet, K. M. Ewen, R. Bernhardt: Cytochrome p450 systems – biological variations of electron transport chains. Biochemica et Biophysica Acta 1770 (2007) 330 - 344
[2] Paul R. Ortiz de Montellano. Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes. Chem Rev. 2010 February 10; 110(2): 932
[3] http://www.anaesthetist.com/physiol/basics/metabol/cyp/Findex.htm
[4] T. Laursen, K. Jensen,B. L. Møller: Conformational changes of the NADPH dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450. Biochemica et Biophysica Acta 1814 (2011) 132-138
[5]Moller, B. L. (2010) Plant science. Dynamic metabolons, Science 330, 1328-1329.
[6]Koch, B. M., Sibbesen, O., Halkier, B. A., Svendsen, I., and Moller, B. L. (1995) The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench, Arch Biochem Biophys 323, 177-186.

iGEM Medals Requirements

Bronze

Team registration

Complete Project Summary form

Team Wiki

Present a poster and a talk at the iGEM Jamboree

At least one new submitted and well-characterized standard BioBrick Part or Device.

Silver

Demonstrate that at least one new BioBrick Part of your own design and construction works as expected
Characterize the operation of at least one new BioBrick Part.

Gold

For Gold Medal, either of the following must be checked

Improve an existing BioBrick Part or Device.
Help another iGEM team
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.

Comments or questions to the team? Please mail us at igemcopenhagen@gmail.com

Retrieved from "http://2011.igem.org/Team:Copenhagen/Project"

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-<strong>Cytochrome p450 CYP 79 - the source of CyperMans fungus destroying power</strong>
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-Background information:
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-It has been proposed in the article Dynamic Metabolons [5] that the assembly and disassembly of enzymes complexes, so-called metabolons, may differentiate plant defense responses to insect attack and fungal infection.
-The two CYP79 and CYP71 subfamilies are part of a metabolon complex that shifts between assembly and disassembly, according to interactions with proteins or from specific metabolic demands. The CYP79 subfamily can by itself produce a substance called 4-hydroxyphenyl-acetaldeyde oxime. This substance is generally toxic to fungi and frequently used as chemical fungicides. They function by inhibiting mitochondrial oxidases and thereby promote lipid peroxidation and the production of toxic reactive oxygen species[5].
-The CYP79A1 uses L-Tyrosine as a substrate, which it hydroxylates twice on the amino-group in the presence of NADPH and O2. The products of these reactions are quite unstable and dehydration and decarboxylation will then produce 4-hydroxyphenyl-acetaldeyde oxime. [6] The CYP79B1 isoform is specific for tryptophan and therefore it produces indole 3-acetaldoxime instead, while A2 uses phenylalanine and produces phenylacetaldoxime.
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-The Project: <br>
-Our goal is to express different CYP79’s (more specifically from the subfamilies A1, A2 and B1) in E. Coli, to determine whether they can actually be expressed as working proteins in them. Then our idea is introduce a promoter to the system, which reacts to a specific chemical signal that is sent out by fungi.
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 <strong>Impact factors:</strong>
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 <u>Ecological and economical prospects:</u>
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-Our future aim with our project is to determine whether it would be a viable solution to use an oxime producing agent as a type of fungicide. The best scenario would be if the plants themselves was able to produce the oximes and thus protect themselves and eliminating the need for fungicides.
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-Another use of CyperMan could be in wood protection. Oximes are already used in wood preservatives and paints to protect the wood from fungus and algae. The oximes in use are butanaldoximes, but the oximes produced by CyperMan should have the same effects. By having the cyp under a inducible promoter, induced by a cheap and accesseble compound such as salt water, we could provide a cheap and easy way to protect wood without having to use chemical paint.
-You could simply spray your wooden deck with the bacteria, then with the salt water. The production of oximes would kill fungus, algae and eventually the bacteria itself.
 <br>
 Another future aim with our project is to improve wastewater treatments and thus contribute to a cleaner and uncontaminated drinking water for everyone. With this scientific project, we will move towards this realization, but more research is needed. Our belief is that further research on the use of cytochrome p450 will give a valid and an inexpensive product for use in wastewater treatment plants, thus benefitting the environment and our planet.