| Advances in genetic tools for manipulation of hyperthermophilic microorganisms have opened up possibilities to create novel and potentially disruptive platforms for the production of advanced biofuels and valuable industrial chemical precursors. An 'electrofuel' is a biosynthetic commodity chemical created directly from low-potential electrons (such as hydrogen gas, reduced metals, or electricity) and carbon dioxide. An electrofuel-producing host must be able to extract energy from electron-rich inorganic substrates, fix inorganic carbon dioxide into biologically active intermediates, and catalyze the production of the desired fuel or chemical molecule.;The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle, found exclusively in high temperature archaea such as Metallosphaera sedula, provides a novel and unique biological way to fix inorganic carbon dioxide. M. sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides, and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. The 3HP/4HB cycle consists of 13 enzymes encoded by 16 genes, yet when this work began there were several gaps in our understanding of the basic biochemistry and enzymology of the cycle. Here the biochemical details of the final four enzymes of the 3HP/4HB cycle are revealed.;First, the gene encoding for 4-hydroxybutyrate-CoA ligase was identified, cloned, and fully characterized. Several candidates for this step were suggested by bioinformatic analysis, but none were shown to catalyze this biotransformation. Transcriptomic analysis of cells grown under strict H2-CO 2 autotrophy was consistent with the involvement of two genes, Msed_0406 and Msed_0394. Recombinant versions of these enzymes catalyzed the ligation of CoA to 4HB, with similar affinities for 4HB (Km values of 1.9 and 1.5 mM for Msed_0406 and Msed_0394, respectively), but with different rates (1.69 and 0.22 mumol ∼ min-1 ∼ mg-1 for Msed_0406 and Msed_0394, respectively).;Next all four final enzymes, 4-hydroxybutyrate-CoA ligase (AMP-forming), (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA beta- ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA.;Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. These results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated post-translationally by the protein acetyltransferase (Pat)/Sir2-dependent system.;Genetic tools for chromosomal manipulation in Pyrococcus furiosus , a marine hyperthermophile that grows optimally at 95°C on sugars, has allowed insertion of the 3HP/4HB cycle into a suitable host organism. A temperature-dependent gas-intensive bioreactor system was developed for the detailed characterization of productivity and performance of engineered P. furious strains making 3HP from maltose and CO2 with the first three enzymes of the 3HP/4HB pathway. The largest determinant of 3HP productivity was mass transfer of gaseous CO 2 to the medium; higher agitation and greater interfacial area for mass transfer increased 3HP productivity from 5.8 mg/L/hr to 10-21 mg/L/hr. The overall maximum 3HP titer remained the same (170-230 mg/L), however, indicating that some other mechanism determines the production ceiling. Transcriptomic analysis of several engineered P. furious strains producing 3HP or 4HB revealed that stress responses to constitutively expressed heterologous genes are present at 95°C but not at 73°C. Further analysis suggests that redox balancing will be crucial for achieving high productivity, which is discussed along with alterative approaches and lessons for future engineering prospects. |
