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Hydrogen is an alternative fuel for transportation vehicles because it is clean, sustainable, and highly flammable. However, the production of hydrogen from lignocellulosic biomass by microorganisms presents challenges. This microbial process involves multiple complex steps, including thermal, chemical, and mechanical treatment of biomass to remove hemicellulose and lignin, as well as enzymatic hydrolysis to solubilize the plant cell walls. These steps not only incur costs but also result in the production of toxic hydrolysates, which inhibit microbial growth. A hyper-thermophilic bacterium of Caldicellulosiruptor bescii can produce hydrogen by decomposing and fermenting plant biomass without the need for conventional pretreatment. It is considered as a consolidated bioprocessing (CBP) microorganism. This review summarizes the basic scientific knowledge and hydrogen-producing capacity of C. bescii. Its genetic system and metabolic engineering strategies to improve hydrogen production are also discussed. Key points: • Hydrogen is an alternative and eco-friendly fuel. • Caldicellulosiruptor bescii produces hydrogen with a high yield in nature. • Metabolic engineering can make C. bescii to improve hydrogen production. [ABSTRACT FROM AUTHOR]

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C5 and C6 sugars. In addition, C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii. [ABSTRACT FROM AUTHOR]

Enhanced biohydrogen production from high loads of unpretreated cattle manure by hyperthermophilic Caldicellulosiruptor bescii. [Display omitted] • First use of raw manure as sole carbon source for hyperthermophilic H 2 production. • Adaptation of C. bescii provided maximum H 2 production yield of 161 mL/g VS added. • Maximum mass solubilization of unpretreated cattle manure was around 73%. • Sparging with N 2 /CO 2 mixture lowers product inhibition impacts and enhances yield. • Carbon balance was complete for fermentation of cattle manure at 75 °C. Caldicellulosiruptor bescii is the most thermophilic cellulolytic bacterium capable of fermenting crystalline cellulose identified to date, and it also has a superior ability to degrade plant biomass without any pretreatment. This study is the first to assess the potential of utilizing unpretreated cattle manure (UCM) as a feedstock for hydrogen (H 2) production by C. bescii at a concentration range between 2.5–50 g volatile solids (VS)/L. At 50 g VS/L UCM concentrations, H 2 production ceased due to inhibition of C. bescii. To alleviate the impacts of inhibition, two strategies were adopted: (i) reduction of H 2 build-up in the reactor headspace via gas sparging and (ii) adaptation of C. bescii to UCM via adaptive laboratory evolution (ALE). The former increased H 2 yield by 47% compared to the control reactors, where no sparging was applied. The latter increased H 2 yield by 142% compared to the control reactors inoculated by the wild type C. bescii. The UCM-adapted C. bescii demonstrated a remarkable H 2 yield of 161.3 ± 1.6 mL H 2 /g VS added at 15 g VS/L. This yield represents a twofold increase compared to the maximum H 2 yield reported in the literature amongst fermentation studies utilizing manure as feed. At 15 g VS/L, around 73% of UCM was solubilized, and the carbon balance indicated that most of the effluent carbon was in the sugar- and acid-form. The remarkable ability of C. bescii to produce H 2 from UCM under non-sterile conditions presents a significant potential for sustainable biohydrogen production from renewable feedstocks. [ABSTRACT FROM AUTHOR]

This paper builds a quantitative thermodynamic model for the microbial hydrolysis process (MHP, which uses Caldicellulosiruptor bescii at 75°C for pre-digestion) for producing biogas from a 5-10% aqueous suspension of dairy manure (naturally buffered near pH 7.8 by ammonium bicarbonate) by anaerobic digestion with a mix of acetoclastic and syntrophic methanogenesis. Standard Gibbs energy changes were calculated for the major reactions in pre-digestion, for reactions producing H2, acetate, and CO2 in the digester, and for methanogenesis reactions in the digester. The available data limit the study to analyzing reactions in the digester to reactions of short-chain volatile fatty acids anions. Results are presented as curves of ΔrxnG (Gibbs energy change) vs. acetate concentration. The H2(aq) concentration must be above 1.2×10-9 M to get significant syntrophic methanogenesis, i.e., for ΔrxnG to be negative. The results show syntrophic methanogenesis of propionate, butyrate, and valerate slows as acetate concentration increases because hydrogen production also decreases, and consequently, biogas production from syntrophic methanogenesis slows as acetate increases. Bicarbonate also inhibits both acetoclastic and syntrophic methanogenesis but is necessary to prevent acidification (souring) of the digester. At identical steady-state conditions, acetoclastic methanogenesis runs about 1.4 times faster than syntrophic methanogenesis. Because syntrophic methanogenesis produces acetate catabolized by acetoclastic methanogens, both types of methanogens are necessary to maximize biogas production. The culture in the digester is predicted to evolve to optimize the ratio of acetoclastic methanogens to syntrophic methanogens, a condition signaled by a constant, low acetate concentration in the digester effluent. Obtaining volatile solids reduction as high as 75% with MHP requires a feedstock with less than 25% lignin and a culture of acetoclastic methanogens and syntrophic methanogens and their symbiotic bacteria.

This paper builds a quantitative thermodynamic model for the microbial hydrolysis process (MHP, which uses Caldicellulosiruptor bescii at 75°C for pre-digestion) for producing biogas from a 5-10% aqueous suspension of dairy manure (naturally buffered near pH 7.8 by ammonium bicarbonate) by anaerobic digestion with a mix of acetoclastic and syntrophic methanogenesis. Standard Gibbs energy changes were calculated for the major reactions in pre-digestion, for reactions producing H2, acetate, and CO2 in the digester, and for methanogenesis reactions in the digester. The available data limit the study to analyzing reactions in the digester to reactions of short-chain volatile fatty acids anions. Results are presented as curves of ΔrxnG (Gibbs energy change) vs. acetate concentration. The H2(aq) concentration must be above 1.2×10-9 M to get significant syntrophic methanogenesis, i.e., for ΔrxnG to be negative. The results show syntrophic methanogenesis of propionate, butyrate, and valerate slows as acetate concentration increases because hydrogen production also decreases, and consequently, biogas production from syntrophic methanogenesis slows as acetate increases. Bicarbonate also inhibits both acetoclastic and syntrophic methanogenesis but is necessary to prevent acidification (souring) of the digester. At identical steady-state conditions, acetoclastic methanogenesis runs about 1.4 times faster than syntrophic methanogenesis. Because syntrophic methanogenesis produces acetate catabolized by acetoclastic methanogens, both types of methanogens are necessary to maximize biogas production. The culture in the digester is predicted to evolve to optimize the ratio of acetoclastic methanogens to syntrophic methanogens, a condition signaled by a constant, low acetate concentration in the digester effluent. Obtaining volatile solids reduction as high as 75% with MHP requires a feedstock with less than 25% lignin and a culture of acetoclastic methanogens and syntrophic methanogens and their symbiotic bacteria. [ABSTRACT FROM AUTHOR]

BACKGROUND: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. RESULTS: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. CONCLUSION: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against p NPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60–80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w / v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector. • The increasing energy demand has intensified the need of renewable energy sources. • A novel cellulose-binding domain containing cellobiohydrolase (CbCBH) cloned from Caldicellulosiruptor bescii. • CbCBH exhibited great thermostability making it a promising candidate for biofuel. • Plackett-Burman model evaluated the saccharification conditions of sugarcane bagasse. • Optimized conditions yielded a remarkable 28.45% saccharification efficiency. [ABSTRACT FROM AUTHOR]

Our current understanding of enzymatic polysaccharide degradation has come from a huge number of in vitro studies with purified enzymes. While this vast body of work has been invaluable in identifying and characterizing novel mechanisms of action and engineering desirable traits into these enzymes, a comprehensive picture of how these enzymes work as part of a native in vivo system is less clear. Recently, several model bacteria have emerged with genetic systems that allow for a more nuanced study of carbohydrate active enzymes (CAZymes) and how their activity affects bacterial carbon metabolism. With these bacterial model systems, it is now possible to not only study a single nutrient system in isolation (i.e., carbohydrate degradation and carbon metabolism), but also how multiple systems are integrated. Given that most environmental polysaccharides are carbon rich but nitrogen poor (e.g., lignocellulose), the interplay between carbon and nitrogen metabolism in polysaccharide-degrading bacteria can now be studied in a physiologically relevant manner. Therefore, in this review, we have summarized what has been experimentally determined for CAZyme regulation, production, and export in relation to nitrogen metabolism for two Gram-positive (Caldicellulosiruptor bescii and Clostridium thermocellum) and two Gram-negative (Bacteroides thetaiotaomicron and Cellvibrio japonicus) polysaccharide-degrading bacteria. By comparing and contrasting these four bacteria, we have highlighted the shared and unique features of each, with a focus on in vivo studies, in regard to carbon and nitrogen assimilation. We conclude with what we believe are two important questions that can act as guideposts for future work to better understand the integration of carbon and nitrogen metabolism in polysaccharide-degrading bacteria. Key points: • Regardless of CAZyme deployment system, the generation of a local pool of oligosaccharides is a common strategy among Gram-negative and Gram-positive polysaccharide degraders as a means to maximally recoup the energy expenditure of CAZyme production and export. • Due to the nitrogen deficiency of insoluble polysaccharide-containing substrates, Gram-negative and Gram-positive polysaccharide degraders have a diverse set of strategies for supplementation and assimilation. • Future work needs to precisely characterize the energetic expenditures of CAZyme deployment and bolster our understanding of how carbon and nitrogen metabolism are integrated in both Gram-negative and Gram-positive polysaccharide-degrading bacteria, as both of these will significantly influence a given bacterium's suitability for biotechnology applications. [ABSTRACT FROM AUTHOR]

Abstract Background Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions—introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra).

Abstract Background Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. Results In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. Conclusion This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.

ABSTRACT Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE 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. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

Abstract Genes responsible for the anaerobic catabolism of benzoate in the thermophilic archaeon Ferroglobus placidus were expressed in the thermophilic lignocellulose-degrading bacterium Caldicellulosiruptor bescii, as a first step to engineering this bacterium to degrade this lignin metabolite. The benzoyl-CoA ligase gene was expressed individually, and in combination with benzoyl-CoA reductase and a putative benzoate transporter. This effort also assessed heterologous expression from a synthetically designed operon whereby each coding sequence was proceeded by a unique C. bescii ribosome binding site sequence. The F. placidicus benzoyl-CoA ligase gene was expressed in C. bescii to produce a full-length protein with catalytic activity. A synthetic 6-gene operon encoding three enzymes involved in benzoate degradation was also successfully expressed in C. bescii as determined by RNA analysis, though the protein products of only four of the genes were detected. The discord between the mRNA and protein measurements, especially considering the two genes lacking apparent protein abundance, suggests variable effectiveness of the ribosome binding site sequences utilized in this synthetic operon. The engineered strains did not degrade benzoate. Although the heterologously expressed gene encoding benzoyl-CoA ligase yielded a protein that was catalytically active in vitro, expression in C. bescii of six benzoate catabolism-related genes combined in a synthetic operon yielded mixed results. More effective expression and in vivo activity might be brought about by validating and using different ribosome binding sites and different promoters. Expressing additional pathway components may alleviate any pathway inhibition and enhance benzoyl-CoA reductase activity.

Abstract Background Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria. Results Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum. Conclusion The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

Abstract Background The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (Tb = 56°C), ideally produced from lignocellulosic biomass. Caldicellulosiruptor bescii (Topt 78°C), an extremely thermophilic fermentative bacterium naturally capable of deconstructing and fermenting lignocellulose, was metabolically engineered to produce acetone. When the acetone pathway construct was integrated into a parent strain containing the bifunctional alcohol dehydrogenase from Clostridium thermocellum, acetone was produced at 9.1 mM (0.53 g/L), in addition to minimal ethanol 3.3 mM (0.15 g/L), along with net acetate consumption. This demonstrates that C. bescii can be engineered with balanced pathways in which renewable carbohydrate sources are converted to useful metabolites, primarily acetone and H2, without net production of its native fermentation products, acetate and lactate. [ABSTRACT FROM AUTHOR]

Abstract Background Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Results Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. Conclusions This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

Abstract Background Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. Results To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. Conclusions We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.

Abstract Background Biogas production from lignocellulosic biomass is generally considered to be challenging due to the recalcitrant nature of this biomass. In this study, the recalcitrance of birch was reduced by applying steam-explosion (SE) pretreatment (210 °C and 10 min). Moreover, bioaugmentation with the cellulolytic bacterium Caldicellulosiruptor bescii was applied to possibly enhance the methane production from steam-exploded birch in an anaerobic digestion (AD) process under thermophilic conditions (62 °C). Results Overall, the combined SE and bioaugmentation enhanced the methane yield up to 140% compared to untreated birch, while SE alone contributed to the major share of methane enhancement by 118%. The best methane improvement of 140% on day 50 was observed in bottles fed with pretreated birch and bioaugmentation with lower dosages of C. bescii (2 and 5% of inoculum volume). The maximum methane production rate also increased from 4-mL CH4/g VS (volatile solids)/day for untreated birch to 9–14-mL CH4/g VS/day for steam-exploded birch with applied bioaugmentation. Bioaugmentation was particularly effective for increasing the initial methane production rate of the pretreated birch yielding 21–44% more methane than the pretreated birch without applied bioaugmentation. The extent of solubilization of the organic matter was increased by more than twofold when combined SE pretreatment and bioaugmentation was used in comparison with the methane production from untreated birch. The beneficial effects of SE and bioaugmentation on methane yield indicated that biomass recalcitrance and hydrolysis step are the limiting factors for efficient AD of lignocellulosic biomass. Microbial community analysis by 16S rRNA amplicon sequencing showed that the microbial community composition was altered by the pretreatment and bioaugmentation processes. Notably, the enhanced methane production by pretreatment and bioaugmentation was well correlated with the increase in abundance of key bacterial and archaeal communities, particularly the hydrolytic bacterium Caldicoprobacter, several members of syntrophic acetate oxidizing bacteria and the hydrogenotrophic Methanothermobacter. Conclusion Our findings demonstrate the potential of combined SE and bioaugmentation for enhancing methane production from lignocellulosic biomass.

Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism. [ABSTRACT FROM AUTHOR]

Background: Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results: Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions—introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions: Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra). [ABSTRACT FROM AUTHOR]

Abstract 20(S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because β-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. β-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α-l-arabinopyranoside moiety in compound Y. Therefore, β-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α-l-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of β-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far.

The extreme thermophile Caldicellulosiruptor bescii solubilizes and metabolizes the carbohydrate content of lignocellulose, a process that ultimately ceases because of biomass recalcitrance, accumulation of fermentation products, inhibition by lignin moieties, and reduction of metabolic activity. Deconstruction of low loadings of lignocellulose (5 g/L), either natural or transgenic, whether unpretreated or subjected to hydrothermal processing, by C. bescii typically results in less than 40% carbohydrate solubilization. Mild alkali pretreatment (up to 0.09 g NaOH/g biomass) improved switchgrass carbohydrate solubilization by C. bescii to over 70% compared to less than 30% for no pretreatment, with two‐thirds of the carbohydrate content in the treated switchgrass converted to acetate and lactate. C. bescii grown on high loadings of unpretreated switchgrass (50 g/L) retained in a pH‐controlled bioreactor slowly purged (τ = 80 hr) with growth media without a carbon source improved carbohydrate solubilization to over 40% compared to batch culture at 29%. But more significant was the doubling of solubilized carbohydrate conversion to fermentation products, which increased from 40% in batch to over 80% in the purged system, an improvement attributed to maintaining the bioreactor culture in a metabolically active state. This strategy should be considered for optimizing solubilization and conversion of lignocellulose by C. bescii and other lignocellulolytic microorganisms. [ABSTRACT FROM AUTHOR]

Background: Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. Results: To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. Conclusions: We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing. [ABSTRACT FROM AUTHOR]

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5 · 10 4 and 2.2·10 6 M −1 for cellotetraose and cellohexaose, respectively. The interaction between CbСВМ28 and amorphous cellulose is best described by a two-binding-site model with the binding constants of 1.5 · 10 5 and 1.9 · 10 5 M ‐1 . In a competitive binding assay in the presence of a 10-fold excess of cellohexaose the binding constant of CbСВМ28 to amorphous cellulose was 1.9 · 10 5 M −1 . A two-binding-site model also better approximates the binding to Avicel with the binding constants of 8.3·10 5 and 3.2 · 10 4 M ‐1 ; while in the presence of cellohexaose, the binding is described by a single-binding-site model with the binding constant of 2.3 · 10 4 M −1 . With CbСВМ28 binding to bacterial crystalline cellulose with a constant of 7.4 · 10 4 M −1 , this is the first report of such a strong binding to crystalline cellulose for a module of family 28. [ABSTRACT FROM AUTHOR]

Background: Biogas production from lignocellulosic biomass is generally considered to be challenging due to the recalcitrant nature of this biomass. In this study, the recalcitrance of birch was reduced by applying steam-explosion (SE) pretreatment (210 °C and 10 min). Moreover, bioaugmentation with the cellulolytic bacterium Caldicellulosiruptor bescii was applied to possibly enhance the methane production from steam-exploded birch in an anaerobic digestion (AD) process under thermophilic conditions (62 °C). Results: Overall, the combined SE and bioaugmentation enhanced the methane yield up to 140% compared to untreated birch, while SE alone contributed to the major share of methane enhancement by 118%. The best methane improvement of 140% on day 50 was observed in bottles fed with pretreated birch and bioaugmentation with lower dosages of C. bescii (2 and 5% of inoculum volume). The maximum methane production rate also increased from 4-mL CH4/g VS (volatile solids)/day for untreated birch to 9-14-mL CH4/g VS/day for steam-exploded birch with applied bioaugmentation. Bioaugmentation was particularly effective for increasing the initial methane production rate of the pretreated birch yielding 21-44% more methane than the pretreated birch without applied bioaugmentation. The extent of solubilization of the organic matter was increased by more than twofold when combined SE pretreatment and bioaugmentation was used in comparison with the methane production from untreated birch. The beneficial effects of SE and bioaugmentation on methane yield indicated that biomass recalcitrance and hydrolysis step are the limiting factors for efficient AD of lignocellulosic biomass. Microbial community analysis by 16S rRNA amplicon sequencing showed that the microbial community composition was altered by the pretreatment and bioaugmentation processes. Notably, the enhanced methane production by pretreatment and bioaugmentation was well correlated with the increase in abundance of key bacterial and archaeal communities, particularly the hydrolytic bacterium Caldicoprobacter, several members of syntrophic acetate oxidizing bacteria and the hydrogenotrophic Methanothermobacter. Conclusion: Our findings demonstrate the potential of combined SE and bioaugmentation for enhancing methane production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

20( S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because β-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. β-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α- l-arabinopyranoside moiety in compound Y. Therefore, β-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α- l-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of β-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far. [ABSTRACT FROM AUTHOR]

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg−1 and 0.31 mg·mL−1, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.

Platycodin D (PD), a major saponin (platycoside) in Platycodi radix (balloon flower root), has higher pharmacological activity than the other major platycosides; however, its content in the plant root is only approximately 10% (w/w) and the productivities of PD by several enzymes are still too low for industrial applications. To rapidly increase the total PD content, the β-glucosidase from Caldicellulosiruptor bescii was used for the deglucosylation of the PD precursors platycoside E (PE) and platycodin D3 (PD3) in the root extract into PD. Under the optimized reaction conditions, the enzyme completely converted the PD precursors into PD with the highest productivity reported so far, increasing the total PD content to 48% (w/w). In the biotransformation process, the platycosides in Platycodi radix were hydrolyzed by four pathways: deapiosylated (deapi)-PE → deapi-PD3 → deapi-PD, PE → PD3 → PD, polygalacin D3 → polygalacin D, and 3″-O-acetyl polygalacin D3 → 3″-O-acetyl polygalacin D.

Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of many these enzymes is unknown. One of such enzymes is a predicted GDSL lipase or esterase encoded by the locus Athe_0553. In this study, this probable esterase named as Cbes-AcXE2 was overexpressed in Escherichia coli. The Ni-NTA affinity purified enzyme exhibited an optimum pH of 7.5 at an optimum temperature of 70 °C. Cbes-AcXE2 hydrolyzed p-nitrophenyl (pNP) acetate, pNP-butyrate, and phenyl acetate with approximately equal efficiency. The specific activity and K for the most preferred substrate, phenyl acetate, were 142 U/mg and 0.85 mM, respectively. Cbes-AcXE2 removed the acetyl group of xylobiose hexaacetate and glucose pentaacetate like an acetyl xylan esterase (AcXE). Bioinformatics analyses suggested that Cbes-AcXE2, which carries an SGNH hydrolase-type esterase domain, is a member of an unclassified carbohydrate esterase (CE) family. Moreover, Cbes-AcXE2 is evolutionarily and biochemically similar to an unclassified AcXE, Axe2, of Geobacillus stearothermophilus. Thus, we proposed a novel family of carbohydrate esterase for both Cbes-AcXE2 and Axe2. [ABSTRACT FROM AUTHOR]

Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7 ± 0.1 mol mol −1 total sugar) and cellobiose (4.8 ± 0.3 mol mol −1 total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3 ± 6.9 L kg −1 (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus . The biochemical methane potential of the fermentation end-products was 322 ± 10 L kg −1 (COD t ). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2 MJ kg −1 (VS). [ABSTRACT FROM AUTHOR]

[Display omitted] Naturally occurring, microbial contaminants were found in plant biomasses from common bioenergy crops and agricultural wastes. Unexpectedly, indigenous thermophilic microbes were abundant, raising the question of whether they impact thermophilic consolidated bioprocessing fermentations that convert biomass directly into useful bioproducts. Candidate microbial platforms for biomass conversion, Acetivibrio thermocellus (basionym Clostridium thermocellum ; T opt 60 °C) and Caldicellulosiruptor bescii (T opt 78 °C), each degraded a wide variety of plant biomasses, but only A. thermocellus was significantly affected by the presence of indigenous microbial populations harbored by the biomass. Indigenous microbial growth was eliminated at ≥75 °C, conditions where C. bescii thrives, but where A. thermocellus cannot survive. Therefore, 75 °C is the thermophilic threshold to avoid sterilizing pre-treatments on the biomass that prevents native microbes from competing with engineered microbes and forming undesirable by-products. Thermophiles that naturally grow at and above 75 °C offer specific advantages as platform microorganisms for biomass conversion into fuels and chemicals. [ABSTRACT FROM AUTHOR]

The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO, and H as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose. [ABSTRACT FROM AUTHOR]

Many glycoside hydrolases involved in deconstruction of cellulose and xylan from the excellent plant cell wall polysaccharides-degrader Caldicellulosiruptor bescii have been cloned and analyzed. However, far less is known about the enzymatic breakdown of mannan, an important component of hemicellulose. We herein cloned, expressed and purified the first β-mannosidase Cb Man2A from C. bescii . Cb Man2A is thermophilic, with an optimal temperature of 80 °C. Cb Man2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with Cb Man5A, an endo-mannanase from the same bacterium, in releasing mannose from β-1,4-mannan. Thus Cb Man2A forms the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how Cb Man2A may assist C. bescii in mannan utilization is presented. In addition, Cb Man2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, Cb Man2A represents an excellent model for investigation of the binding of GH2 β-mannosidases to galactomannan. [ABSTRACT FROM AUTHOR]