Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

Abstract

Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na(+)/H(+) antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na(+) motive force that is used to conserve energy by a Na(+)-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.

Keywords: Thermococcales; anaerobic respiration; archaea; carbon monoxide; energy; hydrogen; hyperthermophile.

FIGURE 1

(A) Schematic representation of the CO oxidizing Codh complex of Thermococcus onnurineus, which conserves energy through an ion gradient. The naming of the Mrp and Mbh subunits is matched to those of the Mrp and Mbh subunits of Pyrococcus furiosus (Schut et al., 2013). (B) Operon structure of the Mrp-Mbh-Codh encoding genes (TON_1017-TON_1031). The gene encoding subunit N is depicted here as we have sequence verified that there is no frameshift in this gene, contrary to the published genome sequence.

FIGURE 2

Growth characteristics of P. furiosus strain Codh in the absence (left) and presence (right) of CO with limiting maltose (0.5 g L^-1) and yeast extract (1 g L^-1). Compounds are represented as follows: blue triangles, CO; dark blue diamonds, cell protein; orange squares, H₂; red circles, acetate. Error bars represent SD, n = 3.

FIGURE 3

Growth characteristics of P. furiosus strain Codh in the absence (left) and presence (right) of CO with limiting yeast extract (1 g L^-1). Compounds are represented as follows: blue triangles, CO; dark blue diamonds, cell protein; orange squares, H₂; red circles, acetate. Error bars represent SD, n = 3.

FIGURE 4

Calculation of cell yield from CO utilization in the presence of minimal fixed carbon (1 g L^-1 yeast extract) using an estimated yield of 0.3 ATP per mol CO oxidized/H₂ produced. Cell growth as represented by cell protein is indicated by blue lines with closed circles showing measured protein and open circles indicating calculated protein. Hydrogen formation is represented by orange lines with closed squares showing measured H₂ (mM) and open squares indicating estimated H₂ calculated from the amount of CO utilized.