The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by \u3e95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost \u3e85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost \u3e90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase

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

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA, Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at ∼90% theoretical yield and titer of 70 g/L. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ∼50%, whereas multiple gene deletions of all annotated alcohol dehydrogenases except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked ADH activity (acetaldehyde-reducing) by 93%

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

追加1. 胃切除術後の鼻腔ゾンデ早期抜去について(シンポジウム 中山式切除術をめぐって,第473回千葉医学会例会,第4回佐藤外科例会)

Additional file 4. Proteomic data of strains wild-type, AG553, AG553, AG601 and LL1210 of Clostridium thermocellum

Imitation of a Pre-Designed Irregular 3D Yarn in Given Fabric Structures

The 3D CAD software has obvious advantages in appearance imitating and geometric structure modeling for fabrics. In contemporary 3D CAD fabric systems, only uniform yarns are involved in studies on fabric geometric structures, due to technological limitations, whereas objectives such as irregular/uneven 3D yarns have not been considered much. As the fabric structure or the central curve of the yarn changes, it is difficult to reflect the changed positions of the effect spots of the pre-designed uneven 3D yarns accordingly. In this paper, a key-point-mapping algorithm between the source yarn and the target curve is proposed to reflect the position change in effect spots when the fabric structure changes. By using the shape-preserving quasi-uniform cubic B-spline curve, a simple 3D irregular source yarn is designed using key points and setting their corresponding base cross-sections. The mapping is based on the principle that the lengths of the curve between the key points and the contours of the corresponding base cross-sections of the source yarn remain unchanged. Finally, the control grid of the new 3D yarn in the fabric structure is automatically generated. According to the examples and error analysis, the mapping technique can be applied to arbitrary given fabric structures, and the effect spots of the irregular 3D yarn are reasonably distributed as expected

Finite Element Analysis of Grouting Compactness Monitoring in a Post-Tensioning Tendon Duct Using Piezoceramic Transducers

With the development of the post-tensioning technique, prestressed concrete structures have been widely used in civil engineering. To ensure the long-term effectiveness of the prestressed tendon, the grouting quality of the tendon duct is one of the important factors. However, it is still a challenge to monitor the grouting quality of post-tensioning tendon ducts, due to the invisibility of the grouting. The authors’ previous work proposed a real-time method that employed a stress wave-based active sensing approach with piezoceramic transducers to monitor the grouting compactness of a Post-Tensioning Tendon Duct (PTTD). To further understand the piezoceramic induced stress wave propagation in the PTTD with different grouting levels, this paper develops a two-dimensional finite element model for monitoring the grouting compactness of the tendon duct with a piezoceramic transducer. A smart aggregate (SA) developed to utilize one Lead Zirconate Titanate (PZT) transducer with marble protection is installed in the center location of the tendon duct as an actuator. Two PZT patches are bonded on the bottom and top surface of the tendon duct as the sensors. The analysis results show that the finite element analysis results are in good agreement with the experimental results, which demonstrates that the finite element analysis is feasible and reliable. For the top half of the specimen, not much stress wave could be detected before the full grouting level, except for negligible signals that may propagate through the walls of the tendon duct. When the tendon duct grouting is at 100%, the stress wave propagates to the top of the specimen, and the displacements are symmetric in both left-right and top-bottom directions before the stress waves reach the boundary. The proposed two-dimensional finite element model has the potential to be implemented to simulate the stress wave propagation principle for monitoring grouting compaction of the post-tensioning tendon duct

Crack Detection of FRP-Reinforced Concrete Beam Using Embedded Piezoceramic Smart Aggregates

In this paper, the authors present a stress wave-based active sensing method to detect the crack in FRP-reinforced concrete beams. The embedded smart aggregates (SAs), which utilize Lead Zirconate Titanate (PZT) as transducers, are employed in this research to generate and sense the stress wave. Three specimens are involved in the experimental program and each is made of concrete, longitudinal distributed reinforcement, steel stirrups, main bar (FRP bar or steel bar), and four SAs. A pair of SAs installed on the lower part of the main bar and the other pair of SAs mounted on the upper part of main bar are utilized to monitor the crack occurrence and development in the three test specimens. The signals received by the SA sensors are analyzed in both time domain and frequency domain. The wavelet packet energy is used to extract damage features. The applied load−vertical displacement curves of mid-span in the specimen are obtained. Experimental results show the test specimens experience crushing failure when the concrete compression exceeds its compressive strength. Increasing the contact area between FRP bar and concrete can effectively improve the cracking load of the FRP-reinforced concrete beam and reduce the cracking speed and depth of FRP-reinforced concrete beam; on the other hand, increasing the elastic modulus of the main bar can slow down the crack development of concrete on the upper side of the main bar and decrease the displacement of reinforced concrete beam during the loading test process. The research results show that the developed piezoceramic-based active sensing method, though low-cost, can monitor the crack-induced damage and estimate the process of damage degree in real-time, and has potentials to provide an early warning of crack occurrence and development for FRP-reinforced concrete beams

Expression of adhA from different organisms in Clostridium thermocellum

Abstract Background Clostridium thermocellum is a cellulolytic anaerobic thermophile that is a promising candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels such as ethanol. It was previously shown that expressing Thermoanaerobacterium saccharolyticum adhA in C. thermocellum increases ethanol yield.In this study, we investigated expression of adhA genes from different organisms in Clostridium thermocellum. Methods Based on sequence identity to T. saccharolyticum adhA, we chose adhA genes from 10 other organisms: Clostridium botulinum, Methanocaldococcus bathoardescens, Thermoanaerobacterium ethanolicus, Thermoanaerobacter mathranii, Thermococcus strain AN1, Thermoanaerobacterium thermosaccharolyticum, Caldicellulosiruptor saccharolyticus, Fervidobacterium nodosum, Marinitoga piezophila, and Thermotoga petrophila. All 11 adhA genes (including T. saccharolyticum adhA) were expressed in C. thermocellum and fermentation end products were analyzed. Results All 11 adhA genes increased C. thermocellum ethanol yield compared to the empty-vector control. C. botulinum and T. ethanolicus adhA genes generated significantly higher ethanol yield than T. saccharolyticum adhA. Conclusion Our results indicated that expressing adhA is an effective method of increasing ethanol yield in wild-type C. thermocellum, and that this appears to be a general property of adhA genes