Engineering the hyperthermophilic archaeon <i>Pyrococcus furiosus</i> for 1-propanol production

Society relies heavily on chemicals traditionally produced through the refinement of fossil fuels. The conversion of renewable biomass to value-added chemicals by microbes, particularly hyperthermophiles (T_opt ≥80°C), offers a renewable alternative to this traditional approach. Herein, we describe the engineering of the hyperthermophilic archaeon <i>Pyrococcus furiosus</i>, which grows optimally (T_opt) at 100°C, for the conversion of sugar to 1-propanol. This was accomplished by constructing a hybrid metabolic pathway consisting of two native and seven heterologously produced enzymes to convert acetyl-CoA from carbohydrate metabolism to 1-propanol. A total of eleven foreign genes from two other organisms were utilized, one from the thermophilic bacterium <i>Thermoanaerobacter</i> sp. strain X514 and 10 from the thermoacidophilic archaeon <i>Metallosphaera sedula,</i> both of which grow optimally near 70°C. The recombinant <i>P. furiosus</i> strain produced 1-propanol at similar concentrations (up to ~1 mM) when incubated at 75°C to activate the gene products of <i>Thermoanaerobacter</i> sp. strain X514 and <i>M. sedula</i> and by initially incubating at 95°C for <i>P. furiosus</i> growth and then subsequently returning to 75°C to promote 1-propanol formation. Note that 1-propanol was not produced if the culture was grown only at 95°C. This work has the potential for future optimization through harnessing the genome-scale metabolic model of <i>P. furiosus</i> that was used herein to identify engineering targets to increase 1-propanol titer.IMPORTANCEAs petroleum reserves become increasingly strained, the development of renewable alternatives to traditional chemical synthesis becomes more important. In this work, a high-temperature biological system for sugar to 1-propanol conversion was demonstrated by metabolic engineering of the hyperthermophilic archaeon <i>Pyrococcus furiosus</i> (T_opt 100°C). The engineered strain produced 1-propanol by temperature shifting from 75°C to 95°C and then back to 75°C to accommodate the temperature ranges for native and foreign proteins associated with 1-propanol biosynthesis. Genome-scale metabolic modeling informed the carbon and reductant flux in the system, identified potential factors limiting 1-propanol production, and revealed potential optimization targets.