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Team:KU Leuven/Project/Glucosemodel/MeS
From 2013.igem.org
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
Methyl Salicylate - Wetlab
Methyl salicylate is an organic ester also known as wintergreen oil. It is produced by different species of plants. It is converted from chorismate with salicylate as an intermediate. In E. coli, chorismate is used as a precursor for the amino acids phenylalanine, tryptophane and tyrosine. It is produced from erythrose -4- phosphate and phosphoenolpyruvate through the shikimate pathway (Sprenger et al., 2007). Chorismate is also the precursor of isochorismate, which is used for the biosythesis of quinones, siderophores and folic acid (Dosselaere and Vanderleyden, 2001).
The Methyl Salicylate Pathway
The conversion of Ery4P and PEP to DAHP is catalyzed by DAHP synthase, an enzyme which exists in three isoforms, coded by the genes AroF, AroG and AroH. These isoforms are selectively inhibited by tyrosine, phenylalanine and tryptophan, respectively. In E. coli, about 80%, 20% and 1% of the enzyme activities are contributed by the DAHPS phenotypes of the AroG, AroF and AroH products, respectively (Ikeda et al., 2006).
This means that when there is enough phenylalanine produced by the cell, or enough present in the medium, the production of DAHP, and hence chorismate, will stop by inhibiting the Aro genes. The production of phenylalanine will stop as well by inhibition of PheA, coding for chorismate mutase/prephenate dehydratase.
The conversion from chorismate to methyl salicylate starts with a conversion to isochorismate, catalysed by the enzyme isochorismate synthase, encoded by the pchA gene. This isochorismate is directly converted to salicylate by isochorismate pyruvate/lyase, encoded by the pchB gene. This conversion happens immediately, since the pchA and pchB gene form the pchBA operon which is always transcribed to one mRNA. Conversion of salicylate to methyl salicylate is catalysed by S-adenosylmethionine-dependent methyltransferase, encoded by BSMT1 (Gaille et al., 2003).
Figure 1 | The Shikimate Pathway
A Biobrick to start from
The iGEM team of MIT 2006 already constructed a Biobrick encoding the pchBA and BSMT1 genes, so introducing this brick should induce the production of methyl salicylate, since chorismate is a common metabolite in E. coli. The MIT team discovered however that there was almost no methyl salicylate production observed, only when salicylic acid was added to the medium. Our experiments with this Biobrick confirmed this lack of methyl salicylate production. There had to be something wrong with the conversion of chorismate to salicylate. One possibility is that there is something wrong with the enzymes produced by the pchBA genes. Another one is that there is a lack of chorismate present in the cell. After investigating this matter, we believe that the latter one is the most likely.
The Chorismate Problem
Since chorismate is the precursor for three amino acids in E. coli, we believe that there is not much of the chorismate left to be converted to methyl salicylate, since the cell needs to keep producing a steady amount of essential amino acids.
To overcome this problem, we looked at a study by Sun et al. (2011), in which a synthetic pathway was introduced for the production of mandelic acid from chorismate. They achieved this by deleting different genes encoding enzymes that catalyse competing pathways, as well as by introducing a feedback-insensitive DAHP synthase mutant to increase the carbon flow down the shikimate pathway. This last method gave us inspiration to overcome our own problem.
If we look at the pathway in Figure 1, we can conclude that if the amino acids are present in the medium, the conversion of chorismate to these amino acids will be inhibited allosterically, as wel as the production of DAHP. Our plan is to mutate the AroG gene in a manner that the enzymatic function still remains, but that it is insensitive to allosteric inhibition by phenylalanine. We only mutate the AroG gene since this isoform is responsible for 80% of DAHP synthase activity (Hu et al., 2003).
Necessary Mutations
It is proven that a Pro150Leu point mutation is Phe-insensitive. This mutation is used as a positive control in a study by Hu et al. (2003) in which they compare the effects of different mutation in the AroG gene on the specific enzymatic activity. The results showed that a Leu175Asp mutation also lead to a Phe-insensitive enzyme. Leu175 is located at the bottom of the possible inhibitor binding pocket, and it is believed to be a critical residue.
For unknown reasons, L175D mutation showed an increased specific enzymatic activity compared to the wild type. In more recent studies, L175D is mostly used to obtain a Phe-insensitive DAHPS (Lin et al., 2012). That is why we will try to introduce a plasmid containing a L175D mutated AroG gene.
It is also proven that transcription of the normal DAHP synthase gene will be inhibited when phenylalanine is present in the medium, so only the mutated form will be produced. (REFERENCE?)
