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Cambridge, MA, United States

Zheng T.,Dartmouth College | Zheng T.,BioEnergy Science Center | Olson D.G.,Dartmouth College | Olson D.G.,BioEnergy Science Center | And 14 more authors.
Journal of Bacteriology | Year: 2015

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (~50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. © 2015, American Society for Microbiology. Source

Shaw A.J.,Mascoma Corporation | Shaw A.J.,Novogy Inc. | Guss A.,Oak Ridge National Laboratory | Bahl H.,University of Rostock | Lynd L.R.,Mascoma Corporation
Metabolic Engineering | Year: 2014

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 was investigated as a host for n-butanol production. A systematic approach was taken to demonstrate functionality of heterologous components of the clostridial n-butanol pathway via gene expression and enzymatic activity assays in this organism. Subsequently. , integration of the entire pathway in the wild-type strain resulted in n-butanol production of 0.85. g/L from 10. g/L xylose, corresponding to 21% of the theoretical maximum yield. We were unable to integrate the n-butanol pathway in strains lacking the ability to produce acetate, despite the theoretical overall redox neutrality of n-butanol formation. However, integration of the n-butanol pathway in lactate deficient strains resulted in n-butanol production of 1.05. g/L from 10. g/L xylose, corresponding to 26% of the theoretical maximum. © 2013. Source

Deng Y.,Dartmouth College | Deng Y.,BioEnergy Science Center | Olson D.G.,Dartmouth College | Olson D.G.,BioEnergy Science Center | And 9 more authors.
Metabolic Engineering | Year: 2013

In Clostridium thermocellum, a thermophilic anaerobic bacterium able to rapidly ferment cellulose to ethanol, pyruvate kinase (EC is absent based on both the genome sequence and enzymatic assays. Instead, a new pathway converting phosphoenolpyruvate to pyruvate via a three-step pathway involving phosphoenolpyruvate carboxykinase, NADH-linked malate dehydrogenase, and NADP-dependent malic enzyme has been found. We examined the impact of targeted modification of enzymes associated with this pathway, termed the "malate shunt", including expression of the pyruvate kinase gene from Thermoanaerobacterium saccharolyticum, mutation of the phosphoenolpyruvate carboxykinase and deletion of malic enzyme gene. Strain YD01 with exogenous pyruvate kinase, in which phosphoenolpyruvate carboxykinase expression was diminished by modifying the start codon from ATG to GTG, exhibited 3.25-fold higher ethanol yield than the wild-type strain. A second strain, YD02 with exogenous pyruvate kinase, in which the gene for malic enzyme and part of malate dehydrogenase were deleted, had over 3-fold higher ethanol yield than the wild-type strain. © 2012 Elsevier Inc. Source

Novogy Inc. | Date: 2012-10-23

Biofuels and renewable fuels. Energy management services, namely, providing a service that allows customers to purchase energy, namely, electricity, natural gas and renewable energy, at a fixed price. Production, treatment and refinement of fuel, diesel fuel, biofuel and biodiesel fuel for others. Licensing of intellectual property.

Disclosed are genetically engineered organisms, such as yeast and bacteria, that have the ability to metabolize atypical nitrogen sources, such as melamine and cyanamide. Fermentation methods using the genetically engineered organisms are also described. The methods of the invention are robust processes for the industrial bioproduction of a variety of compounds, including commodities, fine chemicals, and pharmaceuticals.

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