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

Review of auditing in e-business.

This project investigates the implications of e-business risks on the audit of financial statements. It also reviews the applicability of local auditing standards in an e-business environment. Business risks, both traditional and e-business model specific, are studied in detail and their implications on audit discussed. The adequacy of local auditing standards is also examined in comparison to foreign standards. Deficiencies in local standards are highlighted and solutions proposed

Acetabular Reconstruction with Reinforcement Ring and Morsellised Graft: Technique and Medium-term Result

Background: Acetabular bone defects are commonly seen in both primary and secondary total hip arthroplasty, creating difficulties in restoring anatomical hip centres, which results in high mechanical failure rate. Methods: Total hip arthroplasty with acetabular reinforcement rings were performed in 18 hips in 18 patients from 1996 to 2011 in United Christian Hospital. Both clinical and radiographical assessment were performed during follow-up. Results: Eight patients died of unrelated diseases with average follow-up of 30.5 months. At the latest follow-up, none of them showed radiographic signs of loosening or migration of implants and none of them required revision surgery. The remaining 10 patients with mean age of 77.9 years (range, 65–88) at the time of operation were followed-up for an average of 67.4 months (range, 11–121). The average Harris hip score was 78.3 (range, 58.5–87). The average vertical and horizontal difference of hip centres was 1.5 mm superiorly (p = 0.431) and 0.4 mm medially (p = 0.619) respectively when postoperative hip centres were compared to their contralateral hips. The average inclination of the polyethylene cup was 47.8 degrees (range, 42–58). There was no evidence of radiographic loosening during our follow-up and none of them required revision surgery. Conclusion: Acetabular reconstruction with the use of acetabular reinforcement rings and morsellised bone grafts showed satisfactory clinical and radiographic results at a medium-term follow-up

Identifying promoters for gene expression in Clostridium thermocellum

A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase (lacZ) and NADPH-alcohol dehydrogenase (adhB) in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB). Keywords: Promoter, Copy number, Structural instability, Rolling circle replication, lacZ, adh

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results. Keywords: Clostridium Thermocellum, Plasmid, adhE, Structural stability, Gene expressio

Que identidade e que culturas profissionais docentes?

Partindo do conceito de identidade e profissionalidade docente, da construção do conhecimento profissional e das especificidades da “ação de ensinar”, esta comunicação visa refletir sobre as práticas e sobre a cultura profissional dos docentes, procurando lançar algumas hipóteses exploratórias sobre a eventual distinção entre docentes vinculados e não vinculados a associações profissionais. Se, por um lado, a heterogeneidade nas formas de trabalhar dos professores, em que a partilha e a colaboração muitas vezes se mantem adstrita aos colegas do mesmo grupo ou área disciplinar, contribui para uma cultura profissional fragmentada; por outro lado, os professores estão integrados em escolas/contextos diferentes com diferentes culturas (e subculturas), também estas diferentes (rituais, rotinas, ethos) que determinam a sua socialização profissional. Perante este quadro, e considerando “cultura” como herança social, que culturas profissionais detêm os professores? E que eventuais diferenças podem existir nas práticas profissionais de docentes tendo em conta o grupo de recrutamento e a vinculação a associações profissionais?info:eu-repo/semantics/publishedVersio

Ethanol tolerance in engineered strains of Clostridium thermocellum

Abstract Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75–80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production

MOESM3 of Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase

Additional file 3. Fermentation product and sugar residules of 10 Clostridium thermocellum colonies overexpressing AdhA and ApPDC