(627f) Engineering a Cell-Free System- Enzyme Fusion for Enhanced Production (Industry Candidate)

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 that product inhibition still remained an issue, with aldehyde conversion decreasing as more isobutanol is produced. To solve this issue, we proposed a substrate channeling method of genetically fusing the two ketoisovaleric acid pathway enzymes together. By carrying out this fusion, diffusional length for the intermediate can be reduced and the local concentration of the intermediate in the presence of the isobutanol producing enzyme will increase. To test this theory, we designed four fusion proteins: with either a rigid or flexible linker and two arrangements of our two pathway enzymes. After creating these fusion constructs and ascertaining their activity, stability measurements were conducted to test the fusions’ temperature, pH, and isobutanol stability. Using these results, optimized cell-free reactions were conducted, both in solution and immobilized, and the results compared to the previous non-fused system. Future work will examine the cofactor recycling enzyme, formate dehydrogenase, and explore if it can also be fused or co-immobilized to the resin.