Catechol Degradation

Our home province, Alberta, has proven oil reserves of 171.3 billion barrels. These reserves make up the second-largest proven crude oil reserve in the world. This is enough oil to meet Canada’s current oil demand for almost 400 years¹. The oil reserves are in the form of tar sands: a sand, clay, water, and bitumen mixture². The bitumen is separated from the sticky tar sand by washing it with hot water. The heated water acquires toxins from the tar sands: resulting in an environmental issue.

Catechol is a toxic organic molecule commonly found in tailings ponds. Furthermore, many other toxic compounds, such as naphthenic acids, can be metabolized into catechol. Pseudomonas putida demonstrates great metabolic diversity and is able to utilize a wide range of carbon sources, including molecules few other organisms can break down³. The Lethbridge 2008 iGEM isolated the xylE gene (BBa_K147002) from P.putida, which codes for catechol 2,3-dioxygenase (xylE). However, the Edinburgh 2008 iGEM team isolated the xylE gene (BBa_K118021) from P.putida, along with a ribosomal binding site (rbs), so we chose to work with this part. This year we have engineered Escherichia coli DH5αto express xylE. xylE rapidly converts catechol into 2-hydroxymuconic semialdehyde (2-HMS). 2-HMS is a non-toxic, bright yellow molecule that can be metalbolized by E.coli DH5α.

Figure 1. Part BBa_K118021 submitted to the Registry of Standard Biological Parts in 2008 by team Edinburgh. This part catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde.

We will then use our lumazine synthase (LS) microcompartment to isolate the xylE protein. The LS has been previously characterized and shown that it is able to encapsulate other molecules. We can target a protein into the LS through selectively mutating five of the interior amino acids of the LS to glutamate. By attaching a positively charged arginine tag to the C-terminus of the protein for targeting you can selectively target the tagged protein into the compartment (Lethbridge 2009 Modeling) (Seebeck et al., 2006). We will then purify the complex and use for application in tailings ponds water. Once we have demonstrated that we are successfully able to isolate the xylE we will then add more enzymes for the bioremediation of the tailings ponds.

Figure 2. Poly-Arginine tag attached to xylE, resulting in the localization of xylE into the microcompartment.

References

1) Government of Alberta. "About the Resource." Alberta's Oil Sands. 2010. Government of Alberta. 25 October 2010.

2) Davies, M. P.; Rice, S. (16–19 January 2001). "An alternative to conventional tailing management - "dry stack" filtered tailings". Proceedings of the Eighth International Conference on Tailings and Mine Waste. Fort Collins, Colorado, US: Balkema. pp. 411–422.

3) Newton C.M. Gomes, Irina A. Kosheleva, Wolf-Rainer Abraham, Kornelia Smalla (2005) Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community. FEMS Microbiology Ecology 54 (1), 21–33.