Pyrococcus furiosus is a hyperthermophilic archaeon that grows at temperatures between 70-103�C (Topt = 100�C) on carbohydrates, converting them to acetate, CO2, and H2. The recent discovery of a naturally competent strain has catalyzed the development of genetic tools for rapid and efficient chromosomal modification of this unique high-temperature organism. A pyrF gene deletion mutant allows for genetic selection and counter-selection based on uracil biosynthesis, which allows for multiple insertions of linear DNA at different chromosomal regions. Early demonstrations of this genetic system included a temperature-dependent lactate production system and an overexpression strain that produced high levels of soluble hydrogenase. P. furiosus has great potential as a microbial production platform for many reasons, not the least of which is the reduced risk of contamination at temperatures above 70�C. Metabolic engineering efforts are currently underway to develop P. furiosus as a non-photosynthetic platform for converting CO2 into butanol using H2 as sole source of reducing power. The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation pathway from the thermoacidophilic archaeon Metallosphaera sedula (Topt = 73�C), along with a butanol synthesis pathway constructed from various high-temperature organisms (Topt = 60-75�C), has been introduced into P. furiosus under control of the S-layer protein (slp) promoter, giving constitutive, high-expression levels. Production of 3HP, a key intermediate in the pathway and a valuable commodity chemical, has already been demonstrated, and the construction of a strain containing the entire pathway is underway. Systems-levels analysis tools are also being developed for strain optimization, including a hybrid DNA microarray for the COM1 strain to evaluate strain performance and the metabolic impacts of foreign genes.Discussed here will be the current status of metabolic engineering efforts with P. furiosus, the complementary use of in vitro and in vivo analysis for strain optimization, and the benefits and challenges associated with high temperature bioprocessing.

Metabolic engineering of the hyperthermophilic marine archaeon <i>Pyrococcus furiosus</i> as a versatile biofuel and chemical production platform

247th National Meeting (2014)

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Metabolic engineering of the hyperthermophilic marine archaeon Pyrococcus furiosus as a versatile biofuel and chemical production platform