Denitrogenation

Why focus on nitrogen bioremediation?

The removal of nitrogen heterocycles from the petroleum tailings ponds is important for both of the main goals of the OSCAR project. Since nitrogen containing compounds lower the quality and stability of fuel (Katzer & Sivasubramanian, 1979) , their presence would be detrimental to the usefulness of the hydrocarbons OSCAR could create. The presence of nitrogen compounds in our products also hinders our environmental goals as they have been shown to undergo radical changes, yielding highly genotoxic byproducts (Xu et al). The combination of these two factors makes the removal of nitrogen very important to the success of OSCAR.

Our model compound

Carbazole was chosen as the model compound to study nitrogen containing heterocycles as it accounts for about 70% of nitrogen by mass in petroleum tailings ponds, as well as the fact that it is considered one of the more difficult to degrade (Morales). A pathway that is capable of degrading carbazole could also have the ability to degrade many other types of nitrogen containing compounds found in the tailings ponds. Although the genes in our pathway are mostly designed for carbazole degradation, 3 other compounds have also been chosen to be tested on due to their unique properties. Pyrollidine was chosen because it is a similar, yet less complex heterocycle with nitrogen embedded in the ring. Cyclohexylamine was chosen to test the system's ability to degrade compounds that have the nitrogen as a substituent of a ring. Finally, 4-PBAH was chosen as it is a complex naphthenic acid that can serve as a model containing two of OSCAR's target compounds.

Why use synthetic biology to accomplish this goal? Why not chemical methods?

The most widely used chemical method for removing nitrogen from fuel sources is called hydrodenitrogenation (HDN). This process is not very efficient, as only 77% of nitrogen containing compounds are actually removed (Zeuthen et al, 2001). It also requires harsh conditions, for example temperatures around 350 deg C and pressures up to 30 Bar. This is mostly because the nitrogen atoms in the ring must be hydrogenated before the carbon-nitrogen bond can be cleaved (Katzer & Sivasubramanian). The input of reactants into this system is also very costly.

How are we going to accomplish this?

The goal was to use enzymes produced by the Pseudomonas genus that are responsible for the conversion of carbazole to cathecol with the hope that they will also be able to act on similar nitrogen heterocycles.

The first enzyme in this pathway is carbazole-1,9-dioxygenase (CarA) which is responsible for selectively cleaving the first C-N bond in a nitrogen containing ring structure (Xu et al). This is a 4 part enzyme coded by 3 genes (2xCarAa, CarAc, and CarAd). It converts carbazole into 2'-aminobiphenyl-2,3-diol. (Morales) (Put diagram here)

After this step the nitrogen is broken from the ring, however it is still attached to the structure. From this point, there are two options for the removal of nitrogen from the structure. The first option is the anthranilate 1,2-dioxygenase enzyme, coded by the Ant operon in the native carbazole degradation pathway used by Pseudomonas (Diaz). While this option may seem the most reliable method, it does come with some limitations. It is designed to act on the substrate anthranilate, which is produced in the native pathway by the actions of the CarB and CarC enzymes, however since our synthetic pathway does not include these genes as they do not directly attack nitrogen there is a chance that this enzyme will not interact with our remaining nitrogen group. To this end, we have also biobricked a deaminase enzyme from Rhodococcus erythropolis that has been shown to selectively cleave the 2nd C-N bond from a variety of nitrogen heterocycles (Kilbane)(Kayser). This allows an alternative pathway for the 2nd step of nitrogen removal, possibly making the system more efficient and should show less substrate specificity than the Ant genes allowing us to degrade a wider range of nitrogen containing rings. (get the good diagram of lower pathway options from kilbane paper).

Our constructs

mostly going to be pictures here

What have we shown so far?

graph of LD2 carbazole degradation. make sure to use data with replicates.

Experimental Design

graphical version of slideshow