471 research outputs found
Elimination of Hydrogenase Active Site Assembly Blocks H2 Production and Increases Ethanol Yield in Clostridium Thermocellum
Background: The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2 , and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. Results: H2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ΔhydGΔech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ΔhydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, Δ hydG and ΔhydGΔech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. Conclusions: The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes
Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism
NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP+. The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity
Recommended from our members
The effect of lattice temperature on surface damage in fused silica optics
We examine the effect of lattice temperature on the probability of surface damage initiation for 355nm, 7ns laser pulses for surface temperatures below the melting point to temperatures well above the melting point of fused silica. At sufficiently high surface temperatures, damage thresholds are dramatically reduced. Our results indicate a temperature activated absorption and support the idea of a lattice temperature threshold of surface damage. From these measurements, we estimate the temperature dependent absorption coefficient for intrinsic silica
Functional Heterologous Expression of an Engineered Full Length Cipa from Clostridium Thermocellum in Thermoanaerobacterium Saccharolyticum
Background: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. Results:We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipAfrom C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. Conclusion: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA . In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose
Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state
The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations
Recommended from our members
High-resolution 3-D imaging of surface damage sites in fused silica with Optical Coherence Tomography
In this work, we present the first successful demonstration of a non-contact technique to precisely measure the 3D spatial characteristics of laser induced surface damage sites in fused silica for large aperture laser systems by employing Optical Coherence Tomography (OCT). What makes OCT particularly interesting in the characterization of optical materials for large aperture laser systems is that its axial resolution can be maintained with working distances greater than 5 cm, whether viewing through air or through the bulk of thick optics. Specifically, when mitigating surface damage sites against further growth by CO{sub 2} laser evaporation of the damage, it is important to know the depth of subsurface cracks below the damage site. These cracks are typically obscured by the damage rubble when imaged from above the surface. The results to date clearly demonstrate that OCT is a unique and valuable tool for characterizing damage sites before and after the mitigation process. We also demonstrated its utility as an in-situ diagnostic to guide and optimize our process when mitigating surface damage sites on large, high-value optics
Recommended from our members
Mitigation of Laser Damage Growth in Fused Silica with a Galvanometer Scanned CO2 Laser
At the National Ignition Facility (NIF) at the Lawrence Livermore National Laboratory (LLNL), mitigation of laser surface damage growth on fused silica using single and multiple CO{sub 2} laser pulses has been consistently successful for damage sites whose lateral dimensions are less than 100 {micro}m, but has not been for larger sites. Cracks would often radiate outward from the damage when a CO{sub 2} pulse was applied to the larger sites. An investigation was conducted to mitigate large surface damage sites using galvanometer scanning of a tightly focused CO{sub 2} laser spot over an area encompassing the laser damage. It was thought that by initially scanning the CO{sub 2} spot outside the damage site, radiating crack propagation would be inhibited. Scan patterns were typically inward moving spirals starting at radii somewhat larger than that of the damage site. The duration of the mitigation spiral pattern was {approx}110 ms during which a total of {approx}1.3 J of energy was delivered to the sample. The CO{sub 2} laser spot had a 1/e{sup 2}-diameter of {approx}200 {micro}m. Thus, there was general heating of a large area around the damage site while rapid evaporation occurred locally at the laser spot position in the spiral. A 30 to 40 {micro}m deep crater was typically generated by this spiral with a diameter of {approx}600 {micro}m. The spiral would be repeated until there was no evidence of the original damage in microscope images. Using this technique, damage sites as large as 300 mm in size did not display new damage after mitigation when exposed to fluences exceeding 22 J/cm{sup 2} at 355 nm, 7.5 ns. It was found necessary to use a vacuum nozzle during the mitigation process to reduce the amount of re-deposited fused silica. In addition, curing spiral patterns at lower laser powers were used to presumably ''re-melt'' any re-deposited fused silica. A compact, shearing interferometer microscope was developed to permit in situ measurement of the depth of mitigation sites
Recommended from our members
In-situ monitoring of surface post-processing in large aperture fused silica optics with Optical Coherence Tomography
Optical Coherence Tomography is explored as a method to image laser-damage sites located on the surface of large aperture fused silica optics during post-processing via CO{sub 2} laser ablation. The signal analysis for image acquisition was adapted to meet the sensitivity requirements for this application. A long-working distance geometry was employed to allow imaging through the opposite surface of the 5-cm thick optic. The experimental results demonstrate the potential of OCT for remote monitoring of transparent material processing applications
Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH
Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc
- …