Degradation of high loads of crystalline cellulose and of unpretreated plant biomass by the thermophilic bacterium Caldicellulosiruptor bescii

Basen, Mirko; Rhaesa, Amanda M.; Kataeva, Irina; Prybol, Cameron J.; Scott, Israel M.; Poole, Farris L.; Adams, Michael W. W.

doi:10.1016/j.biortech.2013.11.024

Cited by 77 publications

(71 citation statements)

References 34 publications

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“…The strains developed here will serve as platforms for further metabolic engineering to increase yield and titer to enable cellulosic biofuel production on an industrial scale. Indeed, significant progress has already been made toward process development in C. bescii using high loads of unpretreated plant biomass (31). Deletion of hydrogenase (pathway 1 in Fig.…”

Section: Resultsmentioning

confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

198

171

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Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production. metabolic engineering | bioenergy and thermophiles

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Section: Resultsmentioning

confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

198

171

View full text Add to dashboard Cite

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“…This was consistent with previous studies with C. bescii that found that lignin and carbohydrate solubilizations were proportional during degradation (31). Caldicellulosiruptor species have not been found to utilize lignin as a carbon source; accordingly, the carbohydrate fraction solubilized from cellulose and switchgrass could be accounted for by primary fermentation products (acetate, lactate, ethanol, and carbon dioxide), cellulose and switchgrass degradation products (acetate and soluble sugars), and Caldicellulosiruptor biomass to greater than 92% carbon balance closure, assuming that CO 2 generation was equal to acetate generation on a molar basis (20) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…The cellulolytic Caldicellulosiruptor species also utilize novel binding proteins (ta pirins) to adhere to plant biomass (19). Several Caldicellulosiruptor species can extensively degrade crystalline cellulose (Avicel), and at least one species, Caldicellulosiruptor bescii, has been reported to utilize unpretreated switchgrass (20,21) and, furthermore, to be metabolically engineered to produce ethanol from plant biomass (22).…”

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confidence: 99%

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization

Zurawski

Conway

Lee

et al. 2015

Appl Environ Microbiol

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c Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO 2 , and H 2 . Caldicellulosiruptor bescii, C. kronotskyensis, and C. saccharolyticus solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (Panicum virgatum) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with C. saccharolyticus, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation. T he genusCaldicellulosiruptor is comprised of Gram-positive, anaerobic bacteria that ferment a variety of simple and complex carbohydrates to primarily H 2 , CO 2 , acetate, and lactate at temperatures at or above 70°C (1). To date, Caldicellulosiruptor species have been isolated globally from terrestrial hot springs and thermal features in locations including the United States (C. owensensis [2,3] and C. obsidiansis [4,5]), Russia (C. bescii [6,7]), C. kronotskyensis [2,8], and C. hydrothermalis [2,8]), New Zealand (C. saccharolyticus [9-11]), and Iceland (C. kristjanssonii [2,12] and C. lactoaceticus [2,13]). Characteristic of Caldicellulosiruptor species are multidomain extracellular and S-layer-associated glycoside hydrolases (GHs) that mediate the microbial conversion of complex carbohydrates (14-16). Sequenced genomes for Caldicellulosiruptor species (2, 4, 6, 10) indicate that some, but not all, encode GH48-containing enzymes (17), and these appear to be essential for crystalline cellulose degradation (18). The cellulolytic Caldicellulosiruptor species also utilize novel binding proteins (ta pirins) to adhere to plant biomass (19). Sever...

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“…Low-osmolarity defined (LOD) medium (22) was prepared from filter sterilized stock solutions. The 50ϫ base salts solution contained 16.5 g of MgCl 2 , 16.5 g of KCl, 12.5 g of NH 4 Cl, 7 g of CaCl 2 ·2H 2 O, and 0.68 g of KH 2 PO 4 per liter. Trace element solution SL-10 is prepared as described previously (23), and the 200ϫ vitamin solution contained the following vitamins (in milligrams) per liter: biotin, 4; folic acid, 4; pyridoxine-HCl, 20; riboflavin, 10; thiamineHCl, 10; nicotinic acid 10; pantothenic acid, 10; vitamin B 12 , 0.2; p-aminobenzoic acid, 10; and lipoic acid, 10.…”

Section: Methodsmentioning

confidence: 99%

“…One of these species, Caldicellulosiruptor bescii, has an optimal growth temperature of 78°C and is the most thermophilic cellulose degrader known to date. It is able to ferment high concentrations of cellulosic feedstock primarily to hydrogen gas, lactate, acetate, and CO 2 (3,4). Species from this genus can degrade cellulose (and also xylan), using novel multidomain glycosyl hydrolases, representing a new paradigm in cellulose conversion by anaerobic thermophiles (2).…”

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confidence: 99%

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

Scott

Rubinstein

Lipscomb

et al. 2015

Appl Environ Microbiol

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bCaldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C 5 and C 6 sugars primarily to hydrogen gas, lactate, acetate, and CO 2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.T hermophilic bacteria of the genus Caldicellulosiruptor are currently under intense investigation due to their ability to decompose lignocellulosic plant biomass anaerobically at high temperature, thereby potentially mitigating costly thermochemical pretreatment steps (1, 2). One of these species, Caldicellulosiruptor bescii, has an optimal growth temperature of 78°C and is the most thermophilic cellulose degrader known to date. It is able to ferment high concentrations of cellulosic feedstock primarily to hydrogen gas, lactate, acetate, and CO 2 (3, 4). Species from this genus can degrade cellulose (and also xylan), using novel multidomain glycosyl hydrolases, representing a new paradigm in cellulose conversion by anaerobic thermophiles (2). Moreover, the recent development of a genetic system for C. bescii creates potential for using this and related species for consolidated biomass processing in the production of liquid fuels (5).Developing metabolic engineering strategies for any microorganism obviously requires an in-depth understanding of their primary metabolism. Evidence that C. bescii may have a completely uncharacterized aspect to its primary redox metabolism came from an analysis of its genome sequence for molybdoenzymes (6). These are present in virtually all forms of life, serving diverse roles in primary metabolism of carbon, nitrogen and sulfur (7). As expected, we found that the C. bescii genome contains genes necessary for the synthesis of the pyranopterin cofactor that coordinates molybdenum (Mo) in such enzymes (7). Accordin...

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Degradation of high loads of crystalline cellulose and of unpretreated plant biomass by the thermophilic bacterium Caldicellulosiruptor bescii

Cited by 77 publications

References 34 publications

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

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