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 GC analyses.

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.

We characterised the MeS BioBrick by means of a smell test as well as characterising this brick with Headspace GC, GC-MS analysis of MeS in ethyl acetate extracts and SDS PAGE analysis.

Figure 5 | Incubate at room temperature for 24 hours

Figure 7 | Incubate at 37 degrees for 8 hours

Figure 6 | Incubate at 37 degrees for 24 hours

Figure 8 | Incubate at room temperature for 8 hours

Headspace gas chromatography (HS-GC) was used for the measurement of MeS production by our BBa_K1060003 brick in a BL21 strain. Samples of 5 ml supernatant (centrifuged and filtersterilized) were collected in 15-ml precooled glass tubes, which were immediately closed and cooled on ice. Samples were then analyzed with a calibrated Autosystem XL gas chromatograph with a headspace sampler (HS40; Perkin-Elmer, Wellesley, Mass.) and equipped with a CP-Wax 52 CB column (length, 50 m; internal diameter, 0.32 mm; layer thickness, 1.2 μm; Chrompack; Varian, Palo Alto, Calif.). Samples were heated for 16 min at 72°C in the headspace autosampler. The injection block and flame ionization detector (FID) temperatures were kept constant at 180 and 250°C, respectively; helium was used as the carrier gas. The oven temperature was 75°C held for 6 min and then increased to 110°C at 25°C min−1 and held at 100°C for 3.5 min. Results were analyzed with Perkin-Elmer Turbochrom Navigator software. MeS (Sigma-Aldrich, M 6752) was used as a standard and was detected at 12.832 minutes post injection (A). Cultures of E. coli BL21 and BL21 harbouring BBa_K1060003 with or without 0.1 mM salicylic acid (SA) supplemented to the medium were also analyzed. No MeS could be detected with the FID in the samples when no SA was added (B), while MeS could be detected when SA was supplemented (C).
The results are shown below:

Similar to our set-up for the EBF synthase protein expression, we tested the MIT biobrick (BBa_J45700) and our novel MeS construct (BBa_K1060003) via protein expression. We transformed our constructs in an E.coli expression strain, grew them at various temperatures (room temperature and 37 degrees celcius) and induced expression with increasing amounts of IPTG. We also added salicylate or chorismate to the growth medium in an attempt to increase MeS production.
The figure shows results obtained with the BBa_J45700 brick. Our biobrick (BBa_K1060003) showed similar results (data not shown). Increasing the amount of IPTG did not influence the protein expression profile (compare lanes a-d or lanes e-h) but we do see some bands in the lanes a-d which we cannot see in lanes e-f (eg a band just above 55 kDa). Nonetheless, this observation can be verified with lower amounts of the protein extracts. Nonetheless, our smell test would suggest that the MeS brick does work. Hence, we need a more sensitive approach to identify the protein production of the MeS brick. Possible approaches would be via classic western blot experiment or a GC-MS set-up.

BBa_K1060000 coding biobrick of DAHP synthase, encoded by the aroG gene from E. coli in the pSB1C3 backbone. The insert is 1053 bp long.

Figure 9 | aroG coding sequence (1053 bp) in pSB1C3 backbone (2070 bp). Digestion confirmation, cut with EcoRI and PstI restriction sites. Sequence confirmed.

BBa_K1060003 generator biobrick. It is a twin of the BBa_J47500 biobrick made by the 2006 MIT team, put into a pSB1C3 backbone. The coding sequence is 3255 bp long.

Figure 10 | Methyl salicylate producing construct (3255 bp) in pSB1C3 backbone (2070 bp). Digestion confirmation, cut with EcoRI and PstI restriction sites. Sequence confirmed.