Part:BBa_K4260032
IsoMo: Isoeugenol Monooxygenase
Type:Coding sequence
Designed by:Claudia Angélica GarcÃa Alonso
Group:iGEM_TecCEM
Isoeugenol monooxygenase is the enzyme responsible for the conversion of isoeugenol into vanillin in a direct conversion. Although there are other metabolic pathways reported for the bioconversion of isoeugenol, it was ideal to find a one-step reaction in order to not saturate the bacteria by the insertion of multiple genes, therefore we concluded that isoeugenol monooxygenase was the best option.
Design
Isoeugenol monooxygenase (IsoMo) from Pseudomonas putidaIE27, reported by Yamada, Okada, Yoshida & Nagasawa in 2008, as “Iso†(GenBank access code: BAF62888.1; this sequence gives the bacteria the ability to grow under the presence of isoeugenol and transforms isoeugenol into vanillin. Besides this, it does not lead to the formation of undesired co-products that could have an inhibitory effect on E. coli, such as vanillic acid or acetaldehyde [1]. Vanillin, on its own, does cause an inhibition on E. coli but at much higher concentrations than isoeugenol [2].
The isoeugenol monooxygenase gene selected is reported by Yamada, Yokada, Oshida & Nagasawa (2008), as “Iso†(GenBank access code: BAF62888.1), because they report that the enzyme does not generate undesired products as acetaldehyde and vanillic acid, that could cause inhibition on the bacteria.
Usage and biology
The active site of the enzyme, according to Ryu, Seo, Park, Ahn, Chong, Sadowsky & Hur (2013) [1], is integrated by residues: H167, H218, H282, H471, E135, E349 & E413. As shown in Fig. 2 it is interiorized.
Another important feature of the IsoMo, is the energy heatmap for IsoMo shows that it has good values of ionic strength in a pH range of 4-8, indicating good tolerance to pH changes (Fig 3.) at most of its amino acids, which can be convenient for further usage of this part, given that the culture of certain microorganisms leads to variations on the pH of the culture; however, it is important to point out that the better value of energy occurs at a pH of 8, which matches information reported in BRENDA [3].
Figure 3 shows that the IsoMo from a pH of 4, to lower values, the ionic strength becomes dependent, that is, if the pH decreases, the ionic strength increases, causing a possible loss of stability due to loss of protein solubility due to a salting-out effect. On the other hand, in figure 4 it can be seen that the isoelectric point of the protein is at a pH of 6 in a concentration range of ionic strength from 0.005 to 0.15 M, which corresponds to the theoretical isoelectric point of 5.71 obtained from the expasy software of Swiss Bioinformatics Resource Portal.
Figure. 4 Charge heatmap of Isoeugenol Monooxygenase, per aminoacid.
Figure 5 is a representative image of the results that can be observed in figure 3, of which areas of the enzyme have positive and which one have negative charges based on the pH of the medium where the enzyme is in contact with.
Figure 6 is a 3D model of the representation of the hydrophobicity of IsoMo, in this image, it can be observed in green color the parts are hydrophobic and the purple is the hydrophilic sites.
Application
For more information please check https://parts.igem.org/Part:BBa_K4260006 .
Biosafety
Although this coding sequence comes from a Pseudomonas bacteria, it is not associated with the pathogenicity of the microorganism itself.
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] 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.
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