(250a) Engineering a Cell-Free System- Enzyme Fusion Scaffold for Biofuel Production

Continuous research effort is being put into alternative energy production, such as biofuels, to reduce our reliance on fossil fuels. One of the stumbling blocks in this research is that production in vivo is adversely affected by alcohol toxicity and product inhibition. A solution to this problem has been previously proposed by our lab: remove the enzymes from the cells, immobilize them on resin, and run a cell-free reaction. Our previous research has yielded favorable isobutanol production compared previous in vivo systems. An issue we encountered was a bottleneck at the aldehyde conversion step, with much of the aldehyde remaining unconverted. To solve this issue, we proposed a substrate channeling method of engineering dockerin fusions of the pathway enzymes that have targeted binding to a cohesion scaffold. The rationale behind carrying out this fusion is that by binding the enzymes in close proximity to each other, the diffusional length for the aldehyde can be reduced and the local concentration of this intermediate in the presence of the isobutanol producing enzyme will increase. This immobilization is also more effective than the previous one as the enzymes will bind to a specific cohesin on the scaffold, instead of randomly binding to the resin. This system can be interchanged for other enzymes fused to these dockerins, providing a modular system that is also open to optimizations to improve production.