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The Ruminiclostridium thermocellum cellulosome system is one of the most efficient cellulase systems in nature. The main hydrolysis product of cellulosic saccharification by R. thermocellum was cellobiose, which served as a strong feedback inhibitor to the cellulosome. In this study, R. thermocellum M3, which directly hydrolyzes cellulosic biomass into monosaccharides, was used to degrade cellobiose to investigate the kinetic characteristics of cellobiose degradation. Moreover, the transcriptomic characteristics of strain M3 under cellobiose and Avicel culture conditions were compared. The results indicated that strain M3 was able to grow and hydrolyze cellobiose under cellobiose concentrations higher than 20 g/L. The best growth performance and glucose production were obtained under 12 g/L cellobiose. Compared with that of Avicel, cellobiose had greater β-glucosidase activity under cellobiose culture, with a maximum glucose yield of 767.97±19.13 mg/g cellobiose. The transcription results indicated that R. thermocellum M3 resisted the feedback inhibition of cellobiose by regulating genes encoding β-glucosidase, cellobiose phosphorylase and ABC transporters. The high glucose yield of strain M3 was closely related to the expression of the bglX gene. In addition, the gene related to the chemotactic pathway of strain M3 also increased its perception and adaptation to cellobiose. [Display omitted] • The cellulosome of R. thermocellum M3 remains active under cellobiose more than 20 g/L. • R. thermocellum hydrolyzed cellobiose related with BGL and non-BGL genes. • BglX was potential responsible for the cellobiose hydrolysis by R. thermocellum. • The findings provide solutions for the biorefined of cellulosic energy and fuels. [ABSTRACT FROM AUTHOR]

Abstract Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75–80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75–80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production. [ABSTRACT FROM AUTHOR]

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C-metabolic flux analysis (13C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13C labeling data were used with 13C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis. • Clostridium thermocellum kinetic model parameterized with wild-type 13C-fluxomics sheds light on wild-type physiology and pathway usage. • Substrate-level regulation relief strategies lead to 13.2 g L-1 predicted improvement in ethanol titer. • K-FIT kinetic parameterization along with enzyme docking simulations provides a way to discover substrate-level regulations. • K-FIT and docking formalisms support the presence of 67 regulations that are previously uknown in Clostridium thermocellum. [ABSTRACT FROM AUTHOR]

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. [ABSTRACT FROM AUTHOR]

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

Carbohydrate‐binding modules of the family 54 (CBM54) are characterized by spontaneous rupture of the peptide bond Asn266‐Ser267 (numbering corresponds to that of laminarinase Lic16A of Ruminiclostridium thermocellum). As a result of processing, two parts are formed noncovalently connected to each other. Here, to gain insights into the functional significance of the internal cleavage, we made modifications of the family‐conserved processing site in CBM54 of Lic16A. We demonstrate that the introduced mutations of residues G264 or S267 to alanine block the hydrolysis. Unprocessed, modified proteins bind insoluble polysaccharides pustulan, chitin, xylan, Avicel, phosphoric acid‐swollen cellulose, and β‐d‐glucan of the yeast cell wall 2–20 times worse than the wild‐type module. The data obtained are the first to demonstrate that processing is important for the functioning of CBM54s. [ABSTRACT FROM AUTHOR]

The recent discovery of `lytic' polysaccharide monooxygenases, copper‐dependent enzymes for biomass degradation, has provided new impetus for the analysis of unusual metal‐ion sites in carbohydrate‐active enzymes. In this context, the CAZY family GH124 endoglucanase from Ruminiclostridium thermocellum contains an unusual metal‐ion site, which was originally modelled as a Ca2+ site but features aspartic acid, asparagine and two histidine imidazoles as coordinating residues, which are more consistent with a transition‐metal binding environment. It was sought to analyse whether the GH124 metal‐ion site might accommodate other metals. It is demonstrated through thermal unfolding experiments that this metal‐ion site can accommodate a range of transition metals (Fe2+, Cu2+, Mn2+ and Ni2+), whilst the three‐dimensional structure and mass spectrometry show that one of the histidines is partially covalently modified and is present as a 2‐oxohistidine residue; a feature that is rarely observed but that is believed to be involved in an `off‐switch' to transition‐metal binding. Atomic resolution (<1.1 Å) complexes define the metal‐ion site and also reveal the binding of an unusual fructosylated oligosaccharide, which was presumably present as a contaminant in the cellohexaose used for crystallization. Although it has not been possible to detect a biological role for the unusual metal‐ion site, this work highlights the need to study some of the many metal‐ion sites in carbohydrate‐active enzymes that have long been overlooked or previously mis‐assigned. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic biomass is one of earth's most abundant resources, and it has great potential for biofuel production because it is renewable and has carbon-neutral characteristics. Lignocellulose is mainly composed of carbohydrate polymers (cellulose and hemicellulose), which contain approximately 75 % fermentable sugars for biofuel fermentation. However, saccharification by cellulases is always the main bottleneck for commercialization. Compared with the enzyme systems of fungi, bacteria have evolved distinct systems to directly degrade lignocellulose. However, most reported bacterial saccharification is not efficient enough without help from additional β-glucosidases. Thus, to enhance the economic feasibility of using lignocellulosic biomass for biofuel production, it will be extremely important to develop a novel bacterial saccharification system that does not require the addition of β-glucosidases. Results: In this study, a new thermophilic bacterium named Ruminiclostridium thermocellum M3, which could directly saccharify lignocellulosic biomass, was isolated from horse manure. The results showed that R. thermocellum M3 can grow at 60 °C on a variety of carbon polymers, including microcrystalline cellulose, filter paper, and xylan. Upon utilization of these substrates, R. thermocellum M3 achieved an oligosaccharide yield of 481.5 ± 16.0 mg/g Avicel, and a cellular β-glucosidase activity of up to 0.38 U/mL, which is accompanied by a high proportion (approximately 97 %) of glucose during the saccharification. R. thermocellum M3 also showed potential in degrading natural lignocellulosic biomass, without additional pretreatment, to oligosaccharides, and the oligosaccharide yields using poplar sawdust, corn cobs, rice straw, and cornstalks were 52.7 ± 2.77, 77.8 ± 5.9, 89.4 ± 9.3, and 107.8 ± 5.88 mg/g, respectively. Conclusions: The newly isolated strain R. thermocellum M3 degraded lignocellulose and accumulated oligosaccharides. R. thermocellum M3 saccharified lignocellulosic feedstock without the need to add β-glucosidases or control the pH, and the high proportion of glucose production distinguishes it from all other known monocultures of cellulolytic bacteria. R. thermocellum M3 is a potential candidate for lignocellulose saccharification, and it is a valuable choice for the refinement of bioproducts. [ABSTRACT FROM AUTHOR]

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic biomass is one of earth's most abundant resources, and it has great potential for biofuel production because it is renewable and has carbon-neutral characteristics. Lignocellulose is mainly composed of carbohydrate polymers (cellulose and hemicellulose), which contain approximately 75 % fermentable sugars for biofuel fermentation. However, saccharification by cellulases is always the main bottleneck for commercialization. Compared with the enzyme systems of fungi, bacteria have evolved distinct systems to directly degrade lignocellulose. However, most reported bacterial saccharification is not efficient enough without help from additional β-glucosidases. Thus, to enhance the economic feasibility of using lignocellulosic biomass for biofuel production, it will be extremely important to develop a novel bacterial saccharification system that does not require the addition of β-glucosidases., Results: In this study, a new thermophilic bacterium named Ruminiclostridium thermocellum M3, which could directly saccharify lignocellulosic biomass, was isolated from horse manure. The results showed that R. thermocellum M3 can grow at 60 °C on a variety of carbon polymers, including microcrystalline cellulose, filter paper, and xylan. Upon utilization of these substrates, R. thermocellum M3 achieved an oligosaccharide yield of 481.5 ± 16.0 mg/g Avicel, and a cellular β-glucosidase activity of up to 0.38 U/mL, which is accompanied by a high proportion (approximately 97 %) of glucose during the saccharification. R. thermocellum M3 also showed potential in degrading natural lignocellulosic biomass, without additional pretreatment, to oligosaccharides, and the oligosaccharide yields using poplar sawdust, corn cobs, rice straw, and cornstalks were 52.7 ± 2.77, 77.8 ± 5.9, 89.4 ± 9.3, and 107.8 ± 5.88 mg/g, respectively., Conclusions: The newly isolated strain R. thermocellum M3 degraded lignocellulose and accumulated oligosaccharides. R. thermocellum M3 saccharified lignocellulosic feedstock without the need to add β-glucosidases or control the pH, and the high proportion of glucose production distinguishes it from all other known monocultures of cellulolytic bacteria. R. thermocellum M3 is a potential candidate for lignocellulose saccharification, and it is a valuable choice for the refinement of bioproducts.