Team:KU Leuven/Project/Glucosemodel/MeS

A Biobrick to start from

The iGEM team of MIT 2006 already constructed a Biobrick (BBa_J45700) 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 (see the smell test in the MeS journal). 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. To tackle the low amount of chorismate in the cell, we have introduced an important enzyme in the chorismate pathway, a DAHP synthase encoded by aroG.

Figure 3 | The original methyl salicylate biobrick BBa_J45700

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 2, 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).

Figure 4 | The different pointmutations of aroG and their effect

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 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).

To functionally test our Methyl salicylate production brick (BBa_K1060003), we prepared 68 microcentrifuge tubes to conduct the blind smell test. This experiment consisted of 4 sets, different in incubation time and temperature. The first set was conducted at room temperature for 24 hours, the second was incubated at 37ºC for 24 hours, the third was done in 37ºC for 8 hours and the last one was carried out in 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 negative control (non-MS producing E. Coli).
We had 11 people participated in the blind test and calculated the percentage of people who smelt MS. Based on the percentage, we can drive the conclusions that the smell was stronger after 24 hours incubation time compared to 8 hours. And incubating the bacteria in 37 degrees seemed to be able to enhance the production with regard to room temperature. What’s more, in the microcentrifuge tubes where 0.1mM or 1mM salicylate was added, the smell became more obvious. In addition, the effect of adding chorismate became clearer after 24 hours. The reason why chorismate requires more time to show the effect may lie in the fact that chorismate is the very first precursor of the methyl salicylate production pathway, whereas the salyciate is the precursor that one step closer to methyl salicylate. Most importantly, our brick worked in terms of producing methyl salicylate.
The results are shown below:

Figure 5 | Incubate at room temperature for 24 hours

Figure 6 | Incubate at 37 degrees for 24 hours

Figure 7 | Incubate at 37 degrees for 8 hours

Figure 8 | Incubate at room temperature for 8 hours

We created the following BioBricks for the methyl salicylate part:

BBa_K1060004 intermediate biobrick consisting of bsmt1 followed by a double terminator. It is in the pSB1C3 backbone and the insert is 1177 bp long. Sequence confirmed.

BBa_K1060005 intermediate biobrick consisting of pchBA followed by a double terminator. It is in the pSB1C3 backbone and the insert is 1839 bp long. Sequence confirmed.

Dosselaere, F., Vanderleyden, J., A metabolic node in action: chorismate-utilizing enzymes in microorganisms, Crit Rev Microbiol 2001;27(2):75-131.
Gaille, C., Reimmann, C., Haas D., Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa, The Journal of Biological Chemistry 2003, 278(19):16893-16898.
Hu, C., Jiang, P., Xu, J., Wu, Y., Huang, W., Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli, J. Basic Microbiol. 43 (2003) 5, 399-406.
Ikeda, M., Towards bacterial strains overproducing L-tryptophan and other aromatics by metabolic engineering, Appl Microbiol Biotechnol 2006, 69:615-626.
Lin, S., Meng, ., Jiang, J., Pang, D., Jones, G., OuYang, H. Ren, L., Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12, International Journal of Biological Macromolecules 51 (2012) 915-919.
Sprenger, G., From scratch to value: engineering Escherichia coli wild type cells to the production of L-phenylalanine and other fine chemicals derived from chorismate, Appl Microbiol Biotechnol 2007, 75:739-749.
Sun, Z., Ning, Y., Liu, L., Liu, Y., Sun, B., Jiang, W., Yang, C., Yang, S., Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid, Microbial Cell Factories 2011, 10:71.