Team:UNSW Australia

ASSEMBLASE

Covalently Co-localising Enzymes in a Modular System

The diffusion of reaction intermediates limits the efficiency of many industrial biocatalytic pathways. The UNSW iGEM team has designed the Assemblase self-assembling scaffold system to counter this by increasing the rate and product yield of such reactions.

The scaffold is a heterohexamer composed of two alpha prefoldin and four beta prefoldin subunits. These subunits have been fused to Spy and Snoop Catchers, which covalently bind to Spy and Snoop Tags fused to enzymes of interest.Proof of principle was established using enzymes from the indole-acetic-acid biosynthesis pathway.

The scaffold specifically and covalently co-localises enzymes in a modular system. As a result, substrate can be channelled between enzymes at a much more efficient rate, thanks to the increased concentration of metabolic intermediates in the immediate surroundings of the enzymes.

Advantages of the scaffold include that prefoldin is highly thermostable and chemically resistant, being a protein derived from thermophilic archaea. This permits our scaffold to be used at high temperatures, allowing for increased kinetic energy in our system and therefore an increased rate of catalysis.

The use of Spy and Snoop Tag/Catchers gives our scaffold modularity, but with low cross-reactivity because of the specificity of the irreversible bonds. Modularity is desirable as it means the scaffold can be easily adapted for use in a range of pathways important in industry, bioremediation, and pharmaceutical synthesis.

The tags are also small, so are unlikely to interfere with enzyme functionality when fused to them.