Novel Enzymatic Conversion of Acetyl-CoA to n-Butanol Using Recombinantly Expressed Proteins From Extreme Thermophiles

Electrofuels are an emerging technology being developed to fulfill the need for a renewable energy source that utilizes the existing liquid fuel infrastructure.  Electrofuel projects aim to use microorganisms to produce liquid fuels from inorganic feedstocks such as carbon dioxide and hydrogen, bypassing many of the shortcomings of current biofuel production methods that rely on photosynthesis. Using a hyperthermophilic archaeon, specifically Pyrococcus furiosus, as the host organism, a synthetic metabolic pathway will be developed where two acetyl-CoA molecules are produced from a single acetyl-CoA.  The second acetyl-CoA will then be converted into n-butanol.  Currently, each enzyme in the pathway is being recombinantly expressed and studied.  The project has been divided into five subpathways.  Sub-pathway 5 (SP5), the focus of my research, converts the surplus acetyl-CoA molecule into n-butanol using a series of enzyme-catalyzed reactions with 5 intermediate products.  Such a pathway has never been developed in a thermophilic organism.  Currently, the conversion mechanism is being developed using 5 enzymes from selected thermophilic organisms.  Like the other sub-pathways, the enzymes composing SP5 have been recombinantly expressed in E. coli and purified.  Various enzyme characteristics, such as kinetics, thermostability, and specificity, are being compared between different enzyme candidates to optimize the process.  This project, and in particular SP5, have far-reaching effects.  Since acetyl-CoA is a common metabolic intermediate, this enzymatic process can be used to produce n-butanol from many living organisms.