Team:Concordia/Pathway

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Application of Scaffold: Prototype Pathway

The Ethanol-to-Acetic-Acid Pathway

In nature, the bacteria Clostridium thermocellum expresses a cellulosome, which is a large protein complex used by certain organisms for the conversion of cellulose into sugars, on the outer membrane of its cell. The cellulosome’s extracellular position is advantageous to the reaction’s efficiency as it is thus unnecessary for the large cellulose substrate to be transported into the cell for the breakdown to occur.

The arrangement of the cellulosome on the outer part of the bacterium is very elegant and allows the enzymes to organize themselves in a useful way so they may carry out the complex metabolic process of breaking down cellulose. What if we could take advantage of this efficient method of arrangement of proteins to carry out different metabolic processes? This is one of the great features of our scaffold.

For our work, we wanted to demonstrate the usefulness and immediate practicality of the scaffold in the creation and implementation of metabolic pathways. The enzymes that carry out sequential processes in these pathways can act more efficiently if they are in close proximity of each other. The scaffold and enzymes may be engineered via their cohesins and dockerins to make use of this observation, allowing the pathway enzymes to be adjacently displayed on the scaffold. We decided to test a fairly simple metabolic pathway that was easily measurable and that could illustrate the great potential of the scaffold to aid more complex metabolic processes.

Alcohol dehydrogenase is an enzyme widely produced by many organisms that facilitates the interconversion of alcohols into aldehydes or ketones, depending on the type of alcohol that is used as a substrate. Depending on the organism producing this enzyme, it can be used either for the production of useful ketones or aldehydes via the elimination of alcohols that would otherwise be toxic for the cell, or it can be used to ensure a steady supply of oxidized nicotinamide adenine dinucleotide via the fermentation pathway.

Aldehyde dehydrogenase is another enzyme that is commonly found in different organisms. It catalyzes the oxidation of aldehydes into acetic acid, a carboxylic acid. This is useful for many organisms since it helps in the detoxification of aldehydes that are either acquired or produced by the organism.

We modified these two proteins in such a way that they would each have a different dockerin domain. We expressed the modified proteins in E. coli via an expression vector and purified them for further use. At the same time, we engineered a strain of L. lactis to produce an extracellular scaffold containing two different cohesin groups in its structure, sequentially displayed and complementary to the dockerin domains of the proteins we expressed in E. coli.

The rationale behind this manipulation was the creation of an artificial two-step pathway that could demonstrate the efficacy of the scaffold to harbor multi-step mechanisms, and the increased efficiency that this feature would translate into.

To test that the pathway would actually be more efficient when bound on a scaffold we plan to assay for activity of the two-step pathway. We will first carry out the reaction with the two modified enzymes docked onto the scaffold and then with the unmodified enzymes in solution. Since the final product of the engineered reaction would be acetic acid, we will correlated its production with the total enzymatic activity of the proteins in our pathway. As a control, we used media containing the non-engineered variant of the bacterial strain that did not express the scaffold, along with the modified dehydrogenases and the ethylic alcohol.