Thermophilic lignocellulose deconstruction

Blumer-Schuette, Sara E.; Brown, Steven D.; Sander, Kyle B.; Bayer, Edward A.; Kataeva, Irina; Zurawski, Jeffrey V.; Conway, Jason; Adams, Michael W. W.; Kelly, Robert M.

doi:10.1111/1574-6976.12044

Cited by 164 publications

(139 citation statements)

References 502 publications

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“…C. thermocellum produces cellulosomes, which are multiprotein structures consisting of a protein platform (scaffoldins) with attached cellulolytic enzymes. These enzymes are bound to scaffoldins via type 1 cohesion-dockerin domain interactions, and this large complex is anchored to the cell wall by secondary scaffol-dins and type 2 cohesin-dockerin interations (1). In addition, the bacterium produces free enzymes and "cell-free" cellulosomes (2) and it is a candidate for cellulosic ethanol production (3).…”

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confidence: 99%

“…In addition, the bacterium produces free enzymes and "cell-free" cellulosomes (2) and it is a candidate for cellulosic ethanol production (3). Wild-type C. thermocellum can utilize hexose sugars but not pentose sugars as fermentation substrates, despite having eight genes encoding xylanases (1). The organism has characterized ABC transporters for the uptake of cellodextrins ranging from G2 to G5 with one, CbpA, specific for cellotriose and another specific for laminaribiose (4).…”

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confidence: 99%

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LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ␤-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5-to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and ␣-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters. IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

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LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson

Klingeman

Schlachter

et al. 2017

Appl Environ Microbiol

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“…T hermophilic organisms, Clostridium thermocellum in particular, hold great promise for the production of ethanol from lignocellulosic feedstocks (1,2). C. thermocellum is a thermophilic, Gram-positive obligate anaerobe that rapidly consumes cellulose.…”

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Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

et al. 2015

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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 (ϳ50% 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. IMPORTANCEThis work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production by C. thermocellum and T. saccharolyticum. T hermophilic organisms, Clostridium thermocellum in particular, hold great promise for the production of ethanol from lignocellulosic feedstocks (1, 2). C. thermocellum is a thermophilic, Gram-positive obligate anaerobe that rapidly consumes cellulose. Engineered strains of Thermoanaerobacterium saccharolyticum (3), a thermophilic anaerobe that ferments xylan and other sugars derived from biomass, have been shown to produce ethanol at Ͼ50 g/liter, a near-theoretical yield (4). While comparable concentrations of ethanol are tolerated by selected strains of C. thermocellum (5-7), the maximum reported concentration of ethanol produced by this organism is 23.6 g/liter (8) and th...

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“…6,7 Actinobacteria are a phylum of Gram-positive bacteria that are found abundantly in soil. 8 They include some of the most prolific lignocellulose-degrading bacteria.…”

Section: Introductionmentioning

confidence: 99%

Bioinformatic Analysis of Glycoside Hydrolases in the Proteomes of Mesophilic and Thermophilic Actinobacteria

Barabote¹

2017

MOJPB

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Petroleum reserves are rapidly depleting and alternative renewable sources of energy need to be developed to meet the energy demands of the planet. Lignocellulose has been recognized as a highly promising and renewable resource for the development of clean energy. Thermophilic microbes and thermostable enzymes are being sought for biological conversion of lignocellulose into biofuels. The phylum Actinobacteria includes several efficient cellulose-degrading microorganisms. Genomes of several Actinobacteria have been completely sequenced and deposited in public databases, which are a great resource for uncovering new enzymes and targets for biotechnology. We searched the predicted proteomes of 69 Actinobacteria for the homologs of 20 glycoside hydrolase families relevant to lignocellulose degradation and identified 589 glycoside hydrolase homologs. We analyzed (1) the distribution of the glycoside hydrolase homologs across mesophilic and thermophilic Actinobacteria (2), the domain architecture of cellulases (from GH5 and GH6 families) and xylanases (from GH10 and GH11 families) from mesophilic and thermophilic Actinobacteria, and (3) asymmetric amino acid substitutions between mesophilic and thermophilic glycoside hydrolases. Overall, our data provide new insights into the distribution of different glycoside hydrolases in Actinobacteria as well as into the thermostability features of cellulases and xylanases from Actinobacteria. Our findings provide a basis for genetic engineering of glycoside hydrolases as well as new targets for biotechnology. Keywords

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Thermophilic lignocellulose deconstruction

Cited by 164 publications

References 502 publications

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

Bioinformatic Analysis of Glycoside Hydrolases in the Proteomes of Mesophilic and Thermophilic Actinobacteria

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