Metabolic engineering of the hyperthermophilic archaeon Pyrococcus furiosus for the renewable production of biofuels and commodity chemicals
Keller, Matthew William
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The human race depends primarily on fossil fuels for the production of carbon based commodity chemicals and transportation fuels. Plant biomass is the leading feedstock in efforts to renewably produce liquid transportation fuels, but its use at large scales is inefficient and results in similar carbon emissions as traditional gasoline on an energy basis. Microorganisms, with their extremely diverse metabolic abilities, offer a wide range of alternative strategies for producing renewable fuels. One such strategy is to use a metabolically-engineered microbe to direct carbon dioxide into a carbon fixation cycle and directly into a fuel synthesis pathway. Our strategy is to engineer the hyperthermophile Pyrococcus furiosus (Topt 100°C) to express the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation, drive that pathway with molecular hydrogen via the native soluble hydrogenase, and direct the resulting acetyl-CoA to a bacterial butanol or ethanol fermentation pathway. The work here focuses on three parallel goals of the larger objective. The first is to demonstrate the first sub-pathway of the carbon fixation cycle, the second is to assemble a hybrid pathway for butanol production from acetyl-CoA, and the third is to explore multiple gene donors for ethanol production from acetyl-CoA. The first sub-pathway of the carbon fixation cycle generates the key intermediate 3-HP, which is a valuable plastics precursor that is currently produced from petroleum. The insertion of the five genes encoding this three enzyme pathway into P. furiosus resulted in the accumulation of 50 mg/L 3-HP in the medium at 75°C. Under the controlled conditions of an in vitro assay, 3-HP production was demonstrated to be dependent upon H2 and CO2. Since no single butanol pathway sufficiently thermophilic for expression in P. furiosus was known, an artificial pathway was assembled from multiple gene donors. Concentrated cell suspensions were incubated at 60°C and butanol (70 mg/L) was produced from maltose. Unlike 3-HP and butanol, a small amount of ethanol (40 mg/L) is natively produced by P. furiosus at low growth temperatures. The bifunctional AdhE is capable of the sequential reduction of acetyl-CoA to ethanol. Since acetyl-CoA is the intended link between carbon fixation and fuel synthesis, AdhE was inserted into P. furiosus to demonstrate ethanol production from acetyl-CoA. Eight thermophilic bacteria were used as gene donors and a maximum of 200 mg/L ethanol was produced by recombinant P. furiosus. This demonstrates a new functional route of ethanol production from acetyl-CoA that is directly compatible with the overall strategy.