Adjustable Multi-Enzyme to Cell Surface Anchoring Protein

Abstract:

There are a plethora of enzymes that occur in the natural world which perform reactions that could be immensely useful to humans. Unfortunately, the efficiency of some of these reactions may render their applications logistically unrealistic. The cellulosome scaffolding protein produced by Clostridium thermocellum has been shown to significantly increase the efficiency of cellulose degradation. The scaffolding protein can be reduced in size and adapted for the cell surface of Escherichia coli. Different cohesion sites on the new cell surface display protein can also be introduced to allow for attachment of desired enzymes. Future applications would include producing a collection of distinct versions of the scaffolding protein for unique arrangements and concentrations of enzymes, enabling construction of an extra-cellular assembly line for a variety of multi-enzymatic reactions. This would lay the foundation for making previously infeasible applications of reactions possible through increased efficiency.

Inspiration:

Tuberculosis is caused by bacterial infections of Mycobacterium tuberculosis. The discovery of antibiotics and their use in the treatment of tuberculosis is a double-edged sword: while curing the disease in some cases, it can also cause resistant mutants to emerge. Proper treatment of tuberculosis is typically a multi-drug regiment using first line antiTB drugs: isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin (Long 425-428). Although if the regiment is not prescribed correctly or is not followed, due to misinformation or financial problems, the large population of tubercle bacilli can contain naturally drug-resistant mutants and those mutants can become a large percentage of the population (Long 425-428). Tuberculosis is highly contagious and the spread of resistant mutants is causing more and more drug-resistant tuberculosis cases every year (“World Health Organization”).

A tuberculosis lesion within the body can contain 10^7–10^9 bacilli and 10-1000 of those are resistant to only one of the first line anti-TB drugs, but a case of drug-resistant tuberculosis is only when ≥1/100 of the population is resistant. Drug resistance theory is the most widely accepted explanation for why multi- and extensive-resistant tuberculosis strains are emerging. Drug-resistance is due to the selection do pre-existing resistant mutants in the original bacterial population by drug pressure. The drug pressure in the case for tuberculosis is because Mycobacterium tuberculosis produces a mycolic acid, complex fatty acid, biofilm that protects it from the host’s immune system and makes drug delivery extremely difficult. The original idea for our project was to start the first steps towards an anti-mycobacteria microbe capable of breaking down the mycolic acid biofilm around the bacilli and allow drugs and the host’s immune system to get rid of the infection. This proposal meant that fatty acid degradation and an easy implementation of the degrading enzymes needed to be created.

Clostridium thermocellum among other cellulose degrading organisms naturally produce and utilize a scaffolding protein known as the cellulosome. The structure has been shown to significantly increase the efficiency of the organisms’ cellulose degrading enzymes. The structure itself is composed of a number of smaller parts.

• The enzymatic subunits of the cellulosome include a variety of cellulose degrading enzymes which include binding regions know as type 1 dockerin regions.
• The type 1 dockerin regions of these enzymes bind to the type 1 cohesin regions located on the cellulosome scaffoldin protein.
• The scaffoldin also includes a single type 2 dockerin region which binds to a corresponding type 2 cohesin region.
• The type 2 cohesin region is part of a S-layer binding protein and effectively anchors the cellulosome to the surface of the cell.
• The scaffoldin also includes a cellulose binding domain which attaches to the substrate and further increases the efficiency of C. thermocellum’s cellulose degradation process.

There are a couple of characteristics that make the C. thermocellum cellulosome a good candidate for this project.

• The entirety of the cellulosome is located outside the cell. This means that substrates do not have to be taken into the cell before reactions can occur.
• The cellulosome keeps enzymes within close proximity to the cell. This would be ideal for applications in sensitive environments (like another organism).
• The cellulosome significantly increases the efficiency of C. thermocellum’s cellulose degrading enzymes. The system may do the same for other multiple enzyme processes.
• The cohesin dockerin interactions of a given species are specific to that species. The cohesin of one will not bind to the dockerin of another.

However, there are a number of issues that will have to be addressed before the cellulosome can be used in more practical applications.

• The S-layer binding module is not compatible with gram negative bacteria like E. coli. It is unable to bind to the S-layer of E. coli due to the organism’s outer membrane.
• The cellulosome scaffoldin is coded with a larger gene (roughly 7kb long) that is more difficult to work with during PCR and in plasmids.
• The addition of a greater variety of cohesin and dockerin regions (possibly from other organisms) would be necessary to give the user more control over how the enzymatic subunits bind to the scaffoldin.
• The attachment of enzymes of the user’s choice to the scaffoldin will require that said enzymes are made to incorporate the correct cohesin regions.

References:

Chao, Tiffany. "Tuberculosis Becoming More Drug-Resistant Worldwide." ABC News. ABC News Medical Unit, 30 August 2012. Web. 28 Sep 2012. .

Long, Robert. "Drug-resistant tuberculosis." Canadian Medical Association Journal. 163.4 (2000): 425-428. Web. 28 Sep. 2012. .

"Tuberculosis." World Health Organization. N.p., March 2012. Web. 28 Sep 2012. .

Retrieved from "http://2012.igem.org/Team:Missouri_Miners/Project"