Team:Copenhagen/Project/Fungus

The source of CyperMans

fungus destroying power


Cytochrome p450 CYP 79
The Project

The main goal is to standardize different plant CYP79s (A1, A2 and B1) and deliver them as BioBricks to iGEM.

Indtroduction

The plant cytochrome p450 79 are the crucial starting point in the conversion of aminoacids into cyanogenic glycosides important for defense mechanisms in plants. In this biosynthetic pathway the CYP79s and the CYP71 subfamilies are part of a enzyme complex – a so-called metabolon. It has been proposed in the article Dynamic Metabolons [5] that the assembly and disassembly of the metabolons, may differentiate plant defense responses to insect attack and fungal infection.

When isolated, the CYP79 subfamily will produce an intermediate in the biosynthetic pathway, a substance called an oxime. This substance is generally toxic to fungi and is 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]. We will investigate following CYP79s: A1, A2 and B1.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-acetaldehyde oxime. [6] The CYP79B1 isoform is specific for tryptophan and therefore it produces indole 3-acetaldoxime instead, while A2 uses phenylalanine and produces phenylacetaldoxime.

Experimental design

We will utilize our self-made standardized CYP79s in a biological system consisting of an IPTG inducible promoter, a RBS and a double terminator in a vector, all BioBricks characterized in the partsregistry.org. This system will enable us to control the expression of the given CYP, only being synthesized when IPTG is added. The objective is now to express the different plant CYP79s in E.coli, to determine whether they actually can be expressed as working proteins in them. This expression will be documented on a Western blot. If/When the CYPs are expressed; we will make membrane preparations in which the CYP should be embedded. This enables us to utilize the enzymatic activity of the CYP79s to convert aminoacids to oximes by hydroxylation, showed as a band on a TLC plate. The production of oximes is a key step in our project. If we produce the oximes, we would verify that the disassembly of the plant’s metabolons consisting of CYP79s could lead to production of oximes, which wouldn’t be further converted to cyanogenic glycosides. This would be a proof of the concept, that disassembly of CYP metabolons in plants, functions as a switch from the defense against insects to the defense against fungal infection.

Ecological and economical prospects

The future perspective with our project is to study the use of this oxime producing agent.

CyperMan protecting wood:

One important feature is to determine whether it would be a viable solution to use an oxime producing agent as a type of fungicide. Here one of our ideas is to use of our CyperMan 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. The idea is to paint the wood with our CyperMan. By having the CYP under an inducible promoter, induced by a cheap and accessible compound such as salt water, we could provide a cheap and easy way to protect wood without having to use chemical paint. Another use of our CyperMan could be in the industrial synthesis of oximes, thus using the modified bacteria as a factory to synthesize oximes for use in e.g paint and wood. One idea could be to spray the purifed oximes on the wood, where it would kill fungus and algae. This would be very favorable solution since the hydroxylation reactions carried out by the P450 are difficult to perform using conventional organic synthesis.

CyperMan protecting plants:

CyperMan could be the solution to the problem with pests destroying our farmlands. The idea is the express the CYP79 in plants, but only when harmful fungi are present. A thought is to introduce a promoter to the system, which reacts to a specific chemical signal that is sent out by fungi. This will enable the plants themselves to produce the oximes and thus protect themselves and eliminating the need for fungicides. Another dimension in this idea could be to make the expression of the CYP tissue specific, thus furthermore limit the release of oximes on beneficial fungi. One example is to target fungi attacking plant leaves by only expressing CYP79 in this area.

References
[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.