Many industrial biocatalytic pathways are limited in their efficiency by diffusion of reaction intermediates. In order to increase the rate and product yield of such reactions, the UNSW iGEM team has designed the self-assembling Assemblase scaffolding system, and established proof of principle.

Proof of principle was tested on the indole acetic acid biosynthesis pathway, and future plans include testing this scaffold with other more commercially viable pathways such as the Taxol side-chain biosynthesis pathway.

This novel protein 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.

As a consequence of its modular design, the Assemblase scaffold can be easily adapted for use in a range of pathways important in industry, bioremediation, and pharmaceutical synthesis.

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.