Engineering the hyperthermophilic archaeon Pyrococcus furiosus 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 Pyrococcus furiosus, 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 Thermoanaerobacter sp. strain X514 and 10 from the thermoacidophilic archaeon Metallosphaera sedula, both of which grow optimally near 70°C. The recombinant P. furiosus strain produced 1-propanol at similar concentrations (up to ~1 mM) when incubated at 75°C to activate the gene products of Thermoanaerobacter sp. strain X514 and M. sedula and by initially incubating at 95°C for P. furiosus 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 P. furiosus 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 Pyrococcus furiosus (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.