Team:Nagahama/Description

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香蔵庫(KOZOKO)

~This is our new HERO!!~

Project Description

We examined the antibacterial activity of volatile substance to realize Kozoko. The result suggested that volatile substance of the wasabi, rose, garlic and onion have antibacterial properties. We attempted to identify antibacterial volatile by literature search. Wasabi, rose, garlic and onion have Allyl isothiocyanete, geraniol, allicin, lachrymaltory-factor respectively. Synthesis of antibacterial volatile has many complicated things, so the synthesis requires various enzymes and precursor. Further, We researched whether synthesis of antibacterial volatile will succeed or not in E. coli. The result, E. coli synthesize precursor of geraniol (geranyl diphosphate,GPP). We understood that E. coli can synthesize geraniol using one enzyme. So, we designed system that biosynthesizes geraniol in E. coli.
We will transfer
1. geraniol synthase gene (from Ocimium basilicum).
2. isoprenoid pathway such as MEP pathway gene (dxs, ipsD, ispF, idi, ispA) to biosynthesize GPP that is precursor of geraniol.
3. positive transcription factor of AcrAB-TolC efflux pump gene (MarA) to enhance geraniol tolerance and export geraniol out of cells.

Plasmid Map

We use these genes. It is the m-ispA which we improved to synyhesize only GPP not to synthesize FPP. GES derived from Ocimum Basilicum、we let E. coli overexpress transcription factor MarA letting the expression of pump that the film outside exhausts geraniol activate. (The below）MEP strengthening pathway. We can let E. coli make materials GPP of various terpenoids, FPP, GGPP in large quantities by strengthening MEP pathway.

MEP Pathway

Non-mevalonate (MEP) pathway synthesizes isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).

It is the slightly complicated synthetic path way with six intermediary metabolite before E. coli synthesizes IPP and DMAPP.

The pathway synthesizing same IPP and DMAPP includes a mevalonate path way.

The difference of these pathway is two.

The first difference starts material is the acetyl CoA of 3 molecules by the mevalonate pathway.

In contrast, the second starts material is pyruvic acid and Glyceraldehyde 3-phosphate by the MEP pathway.

The second difference.

Many bacteria and chloroplast of plants has the MEP pathway.

E. coli synthesizes IPP and DMAPP to produce terpene and beta-carotene and quinone by MEP pathway.

Thanks

Thank GenScript for synthesizing geraniol synthase gene.

GenScript helped us.

We really appreciate what you've did.

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