Fusion Protein Development for in Vitro Production of Isobutanol

As climate change continues to endanger the environment, the need for greener alternatives to fossil fuels, such as biofuels, becomes stronger. Current research into biobased cellular production of biofuels is hampered by product inhibition and cellular toxicity. Our lab has previously shown that production of isobutanol via in vitro methods, specifically immobilization of cell-free enzymes, is a viable alternative to in vivo production. Product inhibition remained a problem; however, the intermediate aldehyde to alcohol conversion decreased with increasing alcohol titer. Research into intermediate conversion has provided multiple solutions, one of which is the fusion of the pathway enzymes. Fusing the enzymes reduces the diffusional length scale for the intermediate reaction and increases conversion to the product. Utilizing this concept, the two pathway enzymes of the ketoisovaleric acid pathway, ketoacid decarboxylase and alcohol dehydrogenase, were genetically fused with several linkers in several orientations and assayed. An immobilized reaction was then conducted with the highest performing fusion, as well as the cofactor recycling enzyme, formate dehydrogenase (FDH). Future work will examine either fusing FDH to the pathway fusion or co-immobilizing them both on the same resin.