1-04: A new molecular switch for temperature-regulated bioproduct formation in a hyperthermophilic microorganism

Hyperthermophilic microorganisms have great biotechnological potential for bioconversion; however, molecular tools for their genetic manipulation have only been discovered recently. We present here the first heterologous protein expression and the first induction system for protein and bioproduct production in the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100°C. We have engineered P. furiosus to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. A recombinant strain (LAC) was constructed that expresses a gene (ldh) encoding lactate dehydrogenase from the plant biomass-degrading thermophile, Caldicellulosiruptor bescii (temperature optimum 78°C). Expression of the ldh gene was controlled by a “cold-shock” promoter that is up-regulated when P. furiosus cells are transferred from 98°C to 72°C.  At 98°C the LAC strain fermented sugar to produce acetate, carbon dioxide and hydrogen as end-products and lactate was not detected. When the LAC strain was grown at 72°C or cells were rapidly cooled down to 72°C, the bacterial lactate dehydrogenase was expressed, resulting in up to 3 mM lactate being produced. Expression of a gene from a moderately thermophilic microbe in a hyperthermophile at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for biomass degradation and biofuel formation.

This work was supported by the Bioenergy Science Center (BESC) of the Office of Biological and Environmental Research in the DOE Office of Science and by the DOE ARPA-E Electrofuels Program.