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 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.

The scaffold's modularity also means that it can be easily adapted for use in a range of pathways important in industry, bioremediation, and pharmaceutical synthesis. Proof of principle was established using the indole-acetic-acid biosynthesis pathway.

The Assemblase scaffold is a heterohexamer, composed of two alpha prefoldin and four beta prefoldin subunits. These prefoldin subunits have been fused to Spy and Snoop Catchers, which covalently bind to Spy and Snoop Tags fused to enzymes of interest.

The design of the Assemblase scaffold has a number of advantages. Firstly, as prefoldin is a chaperone protein derived from thermophilic archaea, our scaffold is highly thermostable and chemically resistant. 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. We also elected to utilise the Spy/Snoop Catcher-Tag systems because of their modularity and low rate of cross-reactivity. Additionally, this attachment mechanism only requires small tags to be fused onto any enzymes entering the system, and these are unlikely to interfere with enzyme functionality. The attachment of the Spy/Snoop Catchers with Tags is also permanent, and not reversed by boiling or competing peptides.

The Assemblase scaffold is therefore an incredibly useful foundational tool that may be adapted to co-localise a variety of enzymes for substrate channelling, which could have positive implications for a range of potential applications.