12 Search Results

The order Thermoanaerobacterales currently consists of fermentative anaerobic bacteria, including the genus Caldicellulosiruptor. Caldicellulosiruptor are represented by thirteen species; all, but one, have closed genome sequences. Interest in these extreme thermophiles has been motivated not only by their high optimal growth temperatures (≥70°C), but also by their ability to hydrolyze polysaccharides including, for some species, both xylan and microcrystalline cellulose. Caldicellulosiruptor species have been isolated from geographically diverse thermal terrestrial environments located in New Zealand, China, Russia, Iceland and North America. Evidence of their presence in other terrestrial locations is apparent from metagenomic signatures, including volcanic ash in permafrost. Here,more » phylogeny and taxonomy of the genus Caldicellulosiruptor was re-examined in light of new genome sequences. Based on genome analysis of 15 strains, a new order, Caldicellulosiruptorales, is proposed containing the family Caldicellulosiruptoraceae, consisting of two genera, Caldicellulosiruptor and Anaerocellum. Furthermore, the order Thermoanaerobacterales also was re-assessed, using 91 genome-sequenced strains, and should now include the family Thermoanaerobacteraceae containing the genera Thermoanaerobacter, Thermoanaerobacterium, Caldanaerobacter, the family Caldanaerobiaceae containing the genus Caldanaerobius, and the family Calorimonaceae containing the genus Calorimonas. A main outcome of ANI/AAI analysis indicates the need to reclassify several previously designated species in the Thermoanaerobacterales and Caldicellulosiruptorales by condensing them into strains of single species. Comparative genomics of carbohydrate-active enzyme inventories suggested differentiating phenotypic features, even among strains of the same species, reflecting available nutrients and ecological roles in their native biotopes.« less

To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium.

The extremely thermophilic cellulolytic bacterium, Caldicellulosiruptor bescii , degrades plant biomass at high temperatures without any pretreatments and can serve as a strategic platform for industrial applications. The metabolic engineering of C. bescii , however, faces potential bottlenecks in bio-based chemical productions.

Caldicellulosiruptor species are hyperthermophilic, Gram-positive, anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals and materials from plant biomass. Xylooligomers such as xylobiose and xylotriose that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose, are present in C. bescii fermentations after 90 h incubation and removal or breakdown of these types of xylooligomers is crucial to achieve high conversion of plant biomassmore » to product. In previous studies the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here we report that the co-expression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogeneous xylan.« less

Abstract Background Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood ( Populus trichocarpa ) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii . The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previouslymore » reported on the transgenic poplar samples. Results Of the 17 transgenic poplar lines examined with C. bescii , a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation ( R ²  = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii . Conclusions Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii . One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.« less

Abstract The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of plant lignocellulosic biomass to biofuels and bioproducts. To investigate the synergy of enzymes involved and to further improve the ability of C. bescii to degrade cellulose, we introduced CAZymes that act synergistically with the C. bescii exoproteome in vivo and in vitro. We recently demonstrated that the Acidothermus cellulolyticus E1 endo-1,4-β-D-glucanase (GH5) with a family 2 carbohydrate-binding module (CBM) increased the activity of C. bescii exoproteome on biomass, presumably acting in concert with CelA.more » The β-glucanase, GuxA, from A. cellulolyticus is a multi-domain enzyme with strong processive exoglucanase activity, and the cellobiose phosphorylase from Thermotoga maritima catalyzes cellulose degradation acting synergistically with cellobiohydrolases and endoglucanases. We identified new chromosomal insertion sites to co-express these enzymes and the resulting strain showed a significant increase in the enzymatic activity of the exoproteome.« less

Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, called $$ t\overline{a}pirins\ $$, located proximate to the type IV pilus locus. The C-terminal domain of Caldicellulosiruptor kronotskyensis $$ t\overline{a}pirin\ $$ 0844 (Calkro_0844) is structurally unique and has a cellulose binding affinity akin to that seen with family 3 carbohydrate binding modules (CBM3s). Here, full-length and C-terminal versions of $$ t\overline{a}pirins\ $$ from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis(Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five $$ t\overline{a}pirins\ $$ bound to microcrystalline cellulose, switchgrass, poplar, and filter papermore » but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844, and NA10_0869 (from strongly cellulolytic species). Perhaps this relates to their specific needs to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Calkro_0844 and NA10_0869 share a higher degree of amino acid sequence identity (>80% identity) with each other than either does with Athe_1870 (~50%). The levels of amino acid sequence identity of Calhy_0908 and Calkr_0826 to Calkro_0844 were only 16% and 36%, respectively, although the three-dimensional structures of their C-terminal binding regions were closely related. Unlike the parent strain, C. bescii mutants lacking the $$ t\overline{a}pirin\ $$ genes did not bind to cellulose following short-term incubation, suggesting a role in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, $$ t\overline{a}pirins\ $$ likely help scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species.The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called $$ t\overline{a}pirins\ $$, are involved in the way that Caldicellulosiruptor species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.« less

Biological hydrolysis of microcrystalline cellulose is an uncommon feature in the microbial world, especially among bacteria and archaea growing optimally above 70 degrees C (the so-called extreme thermophiles). In fact, among this group only certain species in the genus Caldicellulosiruptor are capable of rapid and extensive cellulose degradation. Four novel multidomain glycoside hydrolases (GHs) from Caldicellulosiruptor morganii and Caldicellulosiruptor danielii were produced recombinantly in Caldicellulosiruptor bescii and characterized. These GHs are structurally organized with two or three catalytic domains flanking carbohydrate binding modules from Family 3. Collectively, these enzymes represent GH families 5, 9, 10, 12, 44, 48, and 74,more » and hydrolyze crystalline cellulose, glucan, xylan, and mannan, the primary carbohydrates in plant biomass. Degradation of microcrystalline cellulose by cocktails of GHs from three Caldicellulosiruptor species demonstrated that synergistic interactions enable mixtures of multiple enzymes to outperform single enzymes, suggesting a community mode of action for lignocellulose utilization in thermal environments.« less

Abstract Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. Inmore » spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.« less

ABSTRACT Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates toC. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(–) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15%more » (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance.« less