Part:BBa_K4260011

Coding sequence for Isoeugenol Monooxygenase: RBS, signal peptide pelB and Iso


Type:Coding sequence

Designed by:Claudia Angélica García Alonso

Group:iGEM_TecCEM

Figure. 1 Design scheme of the coding sequence.

Design

These part contains the coding sequence of Isoeugenol monooxygenase (IsoMo), reported by Yamada, Okada, Yoshida & Nagasawa [1] (gene Iso), and has an approximate size of 54 kDa; these enzyme comes from Pseudomonas putida IE27. This par also has as an RBS for stimulate de translation of the IsoMo enzyme, using an existing part (BBa_B0030)[2], as well as a signal peptide called pelB, because of its codon optimization for Escherichia coli , as it was decided that the IsoMo enzyme would be directed to the periplasm for the application we needed (for more information check out BBa_K4260007), these was also taken for an existing part (BBa_J32015). [3]

pelB signal peptide

Figure. 2 pelB modeling from UniProt.

pelB gene plays a key role in the construct directing the protein export to the bacterial periplasm, where its folding is enhanced by the disulfide bond formation. When it binds the sequence is subsequently removed by a signal peptidase. Thus, the purification shall not affect the activity of the protein, moreover, avoids its proteolysis, due to this site contains a smaller amount of proteins compared to the cytoplasm as well as protease. [4]

Iso: Isoeugenol Monooxygenase

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).

Figure. 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.

Figure. 4 Results of molecular docking, performed with AutodockVina.
A bond of 2.399 Å is formed between isoeugenol and Met350.

Application

For more information about the enzyme and its possible application as a selection marker, please check out BBa:K4260007.BBa:K4260007.

Biosafety

Although this coding sequence comes from a Pseudomona bacteria, it is not associated with the pathogenicity of the microorganism itself.

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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_B0030

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

[4] B. Gao, D. Zhangsun, Y. Hu, Y. Wu, L. Sheng, L. Fang, X. Wu, J. Yu, S. Luo, Toxicon Official