Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Abstract

Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

Fig. 1.

(A) The synthetic operon constructed to express the M. sedula genes encoding E1 (αβγ), E2, and E3 in P. furiosus under the control of P_slp. This includes P. furiosus RBSs from highly expressed genes encoding pyruvate ferredoxin oxidoreductase subunit γ (porγ, PF0971), the S-layer protein (slp, PF1399), and cold-induced protein A (cipA, PF0190). (B) The first three enzymes of the M. sedula 3-HP/4-HB cycle produce the key intermediate 3-HP. E1 is acetyl/propionyl-CoA carboxylase (αβγ, encoded by Msed_0147, Msed_0148, Msed_1375), E2 is malonyl/succinyl-CoA reductase (Msed_0709), and E3 is malonate semialdehyde reductase (Msed_1993). NADPH is generated by P. furiosus soluble hydrogenase 1 (SH1), which reduces NADP with hydrogen gas. (C) The first three enzymes (E1 to E3) are shown in context of the complete 3-HP/4-HP cycle for carbon dioxide fixation by M. sedula showing the three subpathways, SP1 (blue), SP2 (green), and SP3 (red). (D) The horizontal scheme shows the amount of energy (ATP), reductant (NADPH), oxidant (NAD), and coenzyme A (CoASH) required to generate 1 mol acetyl-CoA from 2 mol carbon dioxide.

Fig. 2.

Temperature-dependent production of the SP1 pathway enzymes in P. furiosus strain PF506. (A) Growth of triplicate cultures at 98°C (red circles) and temperature (black line) for the temperature shift from 98°C to 75°C are shown. (B) Specific activity (μmol NADPH oxidized⋅min^–1⋅mg^–1) of the coupled activity of E2+E3 in cell-free extracts from cultures grown at 95°C to a high cell density of 1 × 10⁸ cells/mL and then incubated for 18 h at the indicated temperature. (C) Activities of E1, E2+E3, and E1+E2+E3 after the temperature shift to 75°C for the indicated period (Fig. S4). The activities of a cell-free extract of autotrophically grown M. sedula cells is also shown (labeled Msed). The specific activities are E1+E2+E3-coupled assay with acetyl-CoA and bicarbonate (blue), E2+E3-coupled assay with malonyl-CoA (red), and E2 with succinyl-CoA (green) as substrates. (D) Temperature dependence of the coupled activity of E2+E3 (blue circles) in the cell-free extracts after induction at 72°C for 16 h. The activity of P. furiosus glutamate dehydrogenase in the same cell-free extracts is also shown (red squares).

Fig. 3.

3-HP production by P. furiosus. Cells were grown at 95°C and then incubated at 72°C for 16 h to produce the SP1 enzymes. (A) In vitro 3-HP production from acetyl-CoA performed in triplicate. The sources of the C1 carbon (CO₂ or HCO₃⁻) and reducing equivalents (NADPH or NADP/H₂) are indicated. Rates are expressed as micromoles 3-HP produced⋅min^–1⋅mg^–1. (B) In vivo 3-HP production by whole cells (static) using maltose as the source of acetyl-CoA in the presence of hydrogen gas and bicarbonate using cells grown in a 100-mL sealed bottle without pH control. The P. furiosus strains are MW56 (circles, blue) and COM1 (squares, red). (C) In vivo 3-HP production by whole cells (stirred) of MW56 using maltose as the source of acetyl-CoA (circles, blue) and E2+E3 specific activity of the cell-free extracts (squares, green) using cells grown in a 20 L fermenter with pH control (pH 6.8).