Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex*

Background: Biohydrogen production from formate may aid in efficient chemical and fuel production using H2 as the energy carrier. Results: The hyperthermophile Pyrococcus furiosus was engineered to convert formate to H2. Conclusion: An 18-gene cluster can be inserted into the P. furiosus chromosome for active production of a membrane-bound system. Significance: This work demonstrates the versatility of this model organism for metabolic engineering purposes. Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.