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Full-Text Articles in Engineering
Characterization Of Xylan Utilization And Discovery Of A New Endoxylanase In Thermoanaerobacterium Saccharolyticum Through Targeted Gene Deletions, Kara K. Podkaminer, Adam M. Guss, Heather L. Trajano, David A. Hogsett, Lee R. Lynd
Characterization Of Xylan Utilization And Discovery Of A New Endoxylanase In Thermoanaerobacterium Saccharolyticum Through Targeted Gene Deletions, Kara K. Podkaminer, Adam M. Guss, Heather L. Trajano, David A. Hogsett, Lee R. Lynd
Dartmouth Scholarship
The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of …
High Ethanol Titers From Cellulose By Using Metabolically Engineered Thermophilic, Anaerobic Microbes, D. Aaron Argyros, Shital A. Tripathi, Trisha F. Barrett, Stephen R. Rogers, Lawrence F. Feinberg, Daniel G. Olson, Justin M. Foden, Bethany B. Miller, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza
High Ethanol Titers From Cellulose By Using Metabolically Engineered Thermophilic, Anaerobic Microbes, D. Aaron Argyros, Shital A. Tripathi, Trisha F. Barrett, Stephen R. Rogers, Lawrence F. Feinberg, Daniel G. Olson, Justin M. Foden, Bethany B. Miller, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza
Dartmouth Scholarship
This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of …
Development Of Pyrf-Based Genetic System For Targeted Gene Deletion In Clostridium Thermocellum And Creation Of A Pta Mutant, Shital A. Tripathi, Daniel G. Olson, D. Aaron Argyros, Bethany B. Miller, Trisha F. Barrett, Daniel M. Murphy, Jesse D. Mccool, Anne K. Warner, Vineet B. Rajgarhia, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza
Development Of Pyrf-Based Genetic System For Targeted Gene Deletion In Clostridium Thermocellum And Creation Of A Pta Mutant, Shital A. Tripathi, Daniel G. Olson, D. Aaron Argyros, Bethany B. Miller, Trisha F. Barrett, Daniel M. Murphy, Jesse D. Mccool, Anne K. Warner, Vineet B. Rajgarhia, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza
Dartmouth Scholarship
We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes …
Identification Of The [Fefe]-Hydrogenase Responsible For Hydrogen Generation In Thermoanaerobacterium Saccharolyticum And Demonstration Of Increased Ethanol Yield Via Hydrogenase Knockout, A. Joe Shaw, David A. Hogsett, Lee R. Lynd
Identification Of The [Fefe]-Hydrogenase Responsible For Hydrogen Generation In Thermoanaerobacterium Saccharolyticum And Demonstration Of Increased Ethanol Yield Via Hydrogenase Knockout, A. Joe Shaw, David A. Hogsett, Lee R. Lynd
Dartmouth Scholarship
Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of …
Microbial Cellulose Utilization: Fundamentals And Biotechnology, Lee R. Lynd, Paul J. Weimer, Willem H. Van Zyl, Isak S. Pretorius
Microbial Cellulose Utilization: Fundamentals And Biotechnology, Lee R. Lynd, Paul J. Weimer, Willem H. Van Zyl, Isak S. Pretorius
Dartmouth Scholarship
Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial …