Identification and Deletion of The Genes Responsible for Hydrogen Production in Thermoanaerobacter Ethanolicus JW200

Background Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. Due to the coupling of hydrogenase activity with fermentation product distribution, understanding hydrogen production of T. ethanolicus, particularly the genes responsible, is valuable for metabolic engineering of the species. Results Utilizing the hydrogenases reported in Thermoanaerobacterium saccharolyticum and Pyrococcus furiosus as templates, BLAST search identified five hydrogenase gene clusters, including two membrane-bound [NiFe] hydrogenases ech and mbh, two cytoplasmic [FeFe] hydrogenases hyd and hydII, and one cytoplasmic [NiFe] hydrogenase shi. The combined deletion of ech, mbh, shi and hydG resulted in a strain that did not produce hydrogen and showed no methyl viologen hydrogenase activity in cell extracts. Strains with deletions of all the hydrogenases except one showed normal hydrogen production. Methyl viologen hydrogenase activity was greatly reduced in all combined deletion strains except the strain with an intact hydG gene. Conclusion High hydrogen production and hydrogenase activities have been observed for T. ethanolicus. Five hydrogenases have been identified. Hydrogen production was eliminated by deleting genes required for all five hydrogenases. Each individual hydrogenase was verified to be capable of producing hydrogen during fermentation, indicating a high degree of redundancy and flexibility in the hydrogenase systems of T. ethanolicus. A large portion of hydrogenase activity is encoded by the [Fe-Fe] hydrogenases.

Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans.

Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation.

QM/MM study of the binding of H2 to MoCu CO dehydrogenase: development and applications of improved H2 van der Waals parameters

The MoCu CO dehydrogenase enzyme not only transforms CO into CO2 but it can also oxidise H2. Even if its hydrogenase activity has been known for decades, a debate is ongoing on the most plausible mode for the binding of H2 to the enzyme active site and the hydrogen oxidation mechanism. In the present work, we provide a new perspective on the MoCu-CODH hydrogenase activity by improving the in silico description of the enzyme. Energy refinement—by means of the BigQM approach—was performed on the intermediates involved in the dihydrogen oxidation catalysis reported in our previously published work (Rovaletti, et al. “Theoretical Insights into the Aerobic Hydrogenase Activity of Molybdenum–Copper CO Dehydrogenase.” Inorganics 7 (2019) 135). A suboptimal description of the H2–HN(backbone) interaction was observed when the van der Waals parameters described in previous literature for H2 were employed. Therefore, a new set of van der Waals parameters is developed here in order to better describe the hydrogen–backbone interaction. They give rise to improved binding modes of H2 in the active site of MoCu CO dehydrogenase. Implications of the resulting outcomes for a better understanding of hydrogen oxidation catalysis mechanisms are proposed and discussed.

Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity

Background The chemolithoautotrophic β-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used. Results Growth, bioenergetic properties, and oxidation–reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L−1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)−1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N′-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH. Conclusions Our results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.

Theoretical Insights into the Aerobic Hydrogenase Activity of Molybdenum–Copper CO Dehydrogenase

The Mo/Cu-dependent CO dehydrogenase from O. carboxydovorans is an enzyme that is able to catalyse CO oxidation to CO 2 ; moreover, it also expresses hydrogenase activity, as it is able to oxidize H 2 . Here, we have studied the dihydrogen oxidation catalysis by this enzyme using QM/MM calculations. Our results indicate that the equatorial oxo ligand of Mo is the best suited base for catalysis. Moreover, extraction of the first proton from H 2 by means of this basic centre leads to the formation of a Mo–OH–Cu I H hydride that allows for the stabilization of the copper hydride, otherwise known to be very unstable. In light of our results, two mechanisms for the hydrogenase activity of the enzyme are proposed. The first reactive channel depends on protonation of the sulphur atom of a Cu-bound cysteine residues, which appears to favour the binding and activation of the substrate. The second reactive channel involves a frustrated Lewis pair, formed by the equatorial oxo group bound to Mo and by the copper centre. In this case, no binding of the hydrogen molecule to the Cu center is observed but once H 2 enters into the active site, it can be split following a low-energy path.

Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant CyanobacteriumAphanothece halophytica

The unicellular halotolerant cyanobacteriumAphanothece halophyticais a potential dark fermentative producer of molecular hydrogen (H2) that produces very little H2under illumination. One factor limiting the H2photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O2) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H2production ofA.halophyticaunder light and dark conditions and on photosynthetic and respiratory activities. The results showed thatA.halophyticatreated with CCCP and DCMU produced H2at three to five times the rate of untreated cells, when exposed to light. The highest H2photoproduction rates,2.26 ± 0.24and3.63 ± 0.26 μmol H2 g−1dry weight h−1, were found in cells treated with 0.5μM CCCP and 50μM DCMU, respectively. Without inhibitor treatment,A.halophyticaincubated in the dark showed a significant increase in H2production compared with cells that were incubated in the light. Only CCCP treatment increased H2production ofA.halophyticaduring dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H2production rate of39.50 ± 2.13 μmol H2 g−1dry weight h−1was found in cells treated with 0.5μM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O2, which promoted bidirectional hydrogenase activity inA.halophyticacells. In addition, only CCCP enhanced the respiration rate, further reducing the O2level. In contrast, DCMU reduced the respiration rate inA.halophytica.