Part:BBa_K4260009

Coding sequence for Isoeugenol Monooxygenase and transcriptional activator: iemR, promoter, signal peptide PelB and Iso

Type:Coding sequence

Designed by:Claudia Angélica García Alonso

Group:iGEM_TecCEM

Fig. 1 Construct design scheme.

Design

This part contains the necessary elements for the transcription and expression of Isoeugenol Monooxygenase, as well as its transcriptional activator. Isoeugenol Monooxygenase (IsoMo) is an enzyme of Pseudomonas putida IE27, reported by Yamada, Okada, Yoshida & Nagasawa [1] (gene Iso), and has an approximate size of 54 kDa. It carries out the direct conversion of isoeugenol to vanillin by a redox reaction. The transcriptional activator is regulated by the same promoter as IsoMo because of the bidirectional arrangement of the promoter and the location of the coding gene in the antisense chain. It was decided to direct it to the periplasm in regard to the application it was needed for (please check out BBa_K4260006), using the already existing part BBa_J32015 [2], because of its codon optimization, specific for E. coli.

Sources, usage and biology

IemR: Transcriptional activator

The transcriptional activator is coded by gene IemR, from Pseudomonas nitroreducens Jin1, (951 bp long) and has a size of about 34.6 kDa. It is specific for the transcription of the Isoeugenol Monooxygenase gene Iem [3]; however, it was decided to employ gene Iso and not IemR because unlike Iem the first one does not lead to the formation of undesired co-products of the reaction like acetaldehyde or vanillic acid [1].

Fig. 2 3D model of IemR, transcriptional activator of Iso.

Promoter region

The promoter region and the transcriptional activator were evaluated by Ryu, Seo, Park, Ahn, Chong, Sadowsky & Hur (2013) [4] as the transcription regulation system for IsoMo. Eugenol and isoeugenol were evaluated as inducers of the transcription of IemR and hence, the expression of Iem. They concluded that isoeugenol was the better activator, and that by inducing the transcription of Iem, and its traduction, more production of isoeugenol monooxygenase was observed in the bacteria.

Promoter region

Isougenol monooxygenase’s tertiary structure model of gene Iso did not exist, therefore it was obtained with I-TASSER, allowing to observe that it is composed mainly of β sheets.

IsoMo is responsible for the direct conversion of isoeugenol to vanillin, requiring only oxygen for the reaction, which can be very convenient when trying to carry this reaction in a prokaryotic cell. It commonly occurs that, when trying to have a prokaryote express too many genes, it gets stressed and does not express them.

The model shown in Figure 3 was useful for the performance of molecular docking, in order to observe its behavior when put in contact with its substrate isoeugenol. The molecular docking was not a blind-type because previously reported residues of the active site were used as reference: H176, H218, H282, H471, E135, E349 & E413 [3]. The final binding score (ΔG°) was of -6.4 and a possible hydrogen bond was shown in methionine 350 (Figure 4).

Fig. 3 3D model of Isoeugenol Monooxygenase, Iso.

This helped confirm the affinity of the enzyme for its substrate, as well as the interaction between the two molecules. After observing these results, it was determined that Iso coding sequence was a good option for the expression of isoeugenol monooxygenase.
	Fig. 4 Results of molecular docking, performed with AutodockVina. A bond of 2.399 Å is formed between isoeugenol and Met350.

Application

In order to know more information about the enzyme and a possible application as a selection marker, please check out BBa:K4260006.

Biosafety

In spite of the two coded proteins being from Pseudomonas bacteria, no hazards were dealt with, because none of them are associated with the pathogenicity of these microorganisms.

References

[1] Yamada, M., Okada, Y., Yoshida, T., & Nagasawa, T. (2008). Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida. Biotechnology letters, 30(4), 665-670.

[2] https://parts.igem.org/Part:BBa_J32015

[3] Ryu, J. Y., Seo, J., Park, S., Ahn, J. H., Chong, Y., Sadowsky, M. J., & Hur, H. G. (2013). Characterization of an isoeugenol monooxygenase (Iem) from Pseudomonas nitroreducens Jin1 that transforms isoeugenol to vanillin. Bioscience, biotechnology, and biochemistry, 120715.

[4] Ryu, J. Y., Seo, J., Ahn, J. H., Sadowsky, M. J., & Hur, H. G. (2012). Transcriptional control of the isoeugenol monooxygenase of Pseudomonas nitroreducens Jin1 in Escherichia coli. Bioscience, biotechnology, and biochemistry, 76(10), 1891-1896.