Future Direction: Cellulase Pairing

            Polysaccharides are a convenient energy storage system for many organisms. In order to use the energy, the organism must break down the polysaccharide chain into smaller mono- and disaccharides. Cellulose is one such polysaccharide, an energy storage form for plants. Glucose monomers link together in long rigid chains to form cellulose.

The plasmid breaks down plant products that are not cellulose, but similar in structure (hemicellulose and pectin). We have three enzymes to accomplish this: xynA, bglS, and yesZ. The endo-1,4-beta-xylanase xynA cleaves xylan polysaccharide chains to form shorter xylan chains. The enzyme yesZ has beta-galactosidase (beta-galacturonidase) activity, cleaving exo-1,4-beta-D-galactans to produce single galactose molecules from RG I Pectin. The endo-1,3-1,4-beta-glucanase (bglS) will cleave internal 1,4 linkages adjacent to 1,3 linkages.

A secretion system (One ring to secrete them all) has been designed to release the enzymes from the bacteria. This mechanism is the central focus of the project and will be the most helpful to other iGEM teams The “One ring to secrete them all” system can be used to secrete any other enzyme/molecule chosen by an iGEM team.

Finally, a safety mechanism is included in the plasmid design. If the plasmid is ejected from the bacteria and picked up by another, the gene producing the toxin will not be suppressed and will kill the cell.

In future experiments, other enzyme-producing genes will be added to break down plant material further. Cellulase genes are first in line, as cellulose is the largest composition of cell wall. The 2008 Edinburgh team designed the genes required to break down cellulose into glucose (http://parts.igem.org/Protein_coding_sequences/Biosynthesis/Cellulose, https://2008.igem.org/Team:Edinburgh). These are an exocellulase (cex from Cellulomonas fimi), an endocellulase (cenA from C. fimi), and a beta-glucosidase (bglX from Cytophaga hutchinsonii). Other enzymes required to fully degrade plant material would be added as well.