Team:KU Leuven/Project/Glucosemodel/MeS

Methyl salicylate (MeS) or wintergreen oil is a plant hormone. It is converted from chorismate with salicylate as an intermediate. In this page we will discuss the pathway that leads up to methyl salicylate and the biobricks that we made. We will end with characterisation of these biobricks using a smell test, an SDS-PAGE and a GC.

The conversion of Ery4P and PEP to DAHP (3-deoxy-D-arabinoheptulosonate 7-phosphate) is catalysed by DAHP synthase, which exists in three isoforms, coded by the genes aroF, aroG and aroH. These isoforms are selectively inhibited by Tyr, Phe and Trp, respectively. In E. coli, about 80% of the enzyme activity by contributed by the DAHPS of aroG(Ikeda et al., 2006).
When there is enough phenylalanine, the production of DAHP, and hence chorismate, will stop by inhibition of the aro genes.
The conversion from chorismate to MeS starts with a conversion to isochorismate, catalysed by isochorismate synthase, encoded by pchA. Isochorismate is directly converted to salicylate by isochorismate pyruvate/lyase, encoded by pchB. This conversion happens immediately, since pchA and pchB form the pchBA operon which is always transcribed to one mRNA. Conversion of salicylate to MeS is catalysed by S-adenosylmethionine-dependent methyltransferase, encoded by bsmt1 (Gaille et al., 2003).

The iGEM team of MIT 2006 already constructed a Biobrick (BBa_J45700) encoding pchBA and bsmt1, for the production of MeS. However, the MIT team only observed MeS production when salicylic acid was added to the medium. Our experiments with this Biobrick confirmed this lack of MeS production (see the smell test below). 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. We have attempted to increase the productivity of the MIT 2006 brick by using stronger ribosome binding sites. Also, we replaced the lac promoter by a TetR-repressible promoter, so the lacI produced in our system doesn't interfere with the transcription of pchBA..

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 MeS.
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 Tyr, Phe or Trp are present in the medium, the conversion of chorismate to these amino acids will be inhibited allosterically, as wel as the production of DAHP (Figure 2). 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 mutations 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 reasons still unknown, 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 (Adhja & Gottesman, 1984). The unmutated version (P150 and L175) of DAHP synthase is shown in the upper two figures, with the inhibitor phenylalanine in the binding pocket on the right. Below it is shown that the two mutations, P150L and L175D, clearly occupy the binding pocket and thereby sterically hinder the binding of phenylalanine.

To functionally test our Methyl salicylate production brick (BBa_K1060003), we prepared 68 microcentrifuge tubes with E.coli expressing the MeS construct to conduct a blind smell test. This experiment consisted of 4 sets, different in incubation time and temperature. The first set was grown at room temperature for 24 hours, the second was incubated at 37ºC for 24 hours, the third was grown at 37ºC for 8 hours and the last set was grown at room temperature for 8 hours. In each set, we inoculated our brick BBa_K1060003 with IPTG, IPTG + salicylate (concentration range from 0.01mM to 1mM), and IPTG + chorismate (concentration range from 0.01mM to 1mM), the same for MIT 2006 brick BBa_J45700. In addition, the same combinations of additives were introduced in parallel for our negative controls (non-MeS producing E. Coli).
We asked 11 people to participate in this blind test and calculated the percentage of people who smelled MeS. We can conclude that the MeS/wintergreen smell was stronger after 24 hours incubation time compared to 8 hours. Growing the bacteria at 37 degrees also produced a more pronounced smell than those at room temperature. What’s more, in the microcentrifuge tubes where 0.1mM or 1mM salicylate was added, the smell became more obvious both after 8 and 24hrs of growth. The positive effect of adding chorismate became noticable after 24 hours. The reason why chorismate requires more time to show an effect may be that chorismate is the very first precursor of the methyl salicylate production pathway, whereas the salicylate is an intermediate closer to the end product. As such, the impact of salicylate on the end product will become noticeable sooner. Most importantly, our brick worked in terms of producing methyl salicylate.
The results are shown below: