Genetic Engineering of Carbon Monoxide-dependent Hydrogen-producing Machinery in <i>Parageobacillus thermoglucosidasius</i>

Abstract

The metabolic engineering of carbon monoxide (CO) oxidizers has the potential to create efficient biocatalysts to produce hydrogen and other valuable chemicals. We herein applied markerless gene deletion to CO dehydrogenase/energyconverting hydrogenase (CODH/ECH) in the thermophilic facultative anaerobe, Parageobacillus thermoglucosidasius. We initially compared the transformation efficiency of two strains, NBRC 107763 T and TG4. We then disrupted CODH, ECH, and both enzymes in NBRC 107763 T. The characterization of growth in all three disruptants under 100% CO demonstrated that both enzymes were essential for CO-dependent growth with hydrogen production in P. thermoglucosidasius. The present results will become a platform for the further metabolic engineering of this organism.

Carbon Monoxide Induced Metabolic Shift in the Carboxydotrophic Parageobacillus thermoglucosidasius DSM 6285

“…Various available metabolic models and fluxes [8,[27][28][29] as well as transcript and proteomic analyses [9,26] of different P. thermoglucosidasius strains have predicted or demonstrated the presence of genes and proteins involved in the complete metabolism of glucose to generate energy and biomass via both the TCA and glyoxylate cycles as well as through various fermentative pathways. Similarly, the basis of the WGS reaction in this bacterium has been demonstrated [10,14,25]. Here, the carboxydotroph P. thermoglucosidasius DSM 6285 was grown under different CO concentrations to further elucidate the mechanism of CO adaptation.…”

“…P. thermoglucosidasius produces biohydrogen (H 2 ) via the water gas shift (WGS) reaction pathway (CO + H 2 O → CO 2 + H 2 ΔG Oʹ = −20 kJ/mol CO) [ 10 ]. It utilises a CO dehydrogenase (CODH)/hydrogen-evolving hydrogenase (HEH; Phc in P. thermoglucosidasius ) complex to oxidise carbon monoxide (CO) to CO 2 plus electrons which are used for reducing the proton (H + ) from H 2 O to H 2 [ 11 , 12 , 13 , 14 ]. Thus far, no empirical evidence exists regarding whether this group 4a hydrogenase, harboured by P. thermoglucosidasius [ 15 ], translocates protons through the cellular membrane to generate energy.…”

“…Diverse CO-oxidizing taxa in the phyla Firmicutes and Proteobacteria , as well as Archaea have been described with various temperature adaptations, including thermophilic and mesophilic forms [ 19 , 20 ]. However, most of the reported CO oxidizers belong to the phylum Firmicutes , which accounts for about 50% of the known diversity and comprises mainly strict anaerobes [ 14 , 21 , 22 ]. Of the various phyla potentially capable of anaerobic CO oxidation, two groups of facultative anaerobes are of particular interest because of their potential application in oxygen-tolerant hydrogen production systems.…”

“…The first group includes marine mesophilic Proteobacteria such as Ferrimonas futtsuensis DSM18154 [ 21 ] and Photobacterium marinum AK15 [ 22 ], whose potential for hydrogen production is based only on genomic analyses [ 23 ]. Conversely, CO oxidation and hydrogen production via the WGS reaction have been demonstrated in the facultatively anaerobic P. thermoglucosidasius [ 14 , 24 ]. This organism grows aerobically until O 2 depletion, whereafter there is a lag phase during which biomass decreases.…”

Not All That Glitters Is Gold: The Paradox of CO-dependent Hydrogenogenesis in Parageobacillus thermoglucosidasius

“…Recent mutagenesis studies ( Adachi et al, 2020 ), involving the knockout of the CODH and hydrogenases loci, individually and collectively, has conclusively shown that the predicted CODH and hydrogenase genes (above) code for the enzymes that catalyze WGS in P. thermoglucosidasius . While the above studies have confirmed the functional role of the coo — phc locus, central questions on the regulation of the process remain unclear.…”