Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus.

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

Coexpression of a ß-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

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 of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-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 exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates.

Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases.IMPORTANCECaldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

Heterologous co-expression of two ß-glucanases and a cellobiose phosphorylase resulted in a significant increase in the cellulolytic activity of the Caldicellulosiruptor bescii exoproteome.

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-ß-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. The ß-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.

Rational development of transformation in Clostridium thermocellum ATCC 27405 via complete methylome analysis and evasion of native restriction-modification systems.

A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction-modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction-modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.

Engineering a spermidine biosynthetic pathway in Clostridium thermocellum results in increased resistance to furans and increased ethanol production.

Polyamines are low molecular weight aliphatic nitrogen compounds found ubiquitously in microorganisms, plants, and animals. Spermidine is a common polyamine that plays a role in stabilizing chromatin, DNA replication, transcription, translation, as well as the regulation of cell growth and apoptosis in eukaryotes. Amines are also associated with defense to a number of environmental stresses including elevated temperature and have been shown to be involved in tolerance to fermentation inhibitors such as furan derivatives and acetic acid in Saccharomyces cerevisiae. While the tolerance and detoxifying mechanisms have been intensively studied, metabolic engineering efforts to construct tolerant and resistant strains have been few. Here we show that exogenously added spermidine confers enhanced tolerance to furans and acetic acid in the Gram-positive bacterium, Clostridium thermocellum. Deletion of the endogenous spermidine synthase resulted in a severe growth defect and hypersensitivity to both furans and acetic acid. Exogenously added spermidine rescued all three phenotypes. Overexpression of the endogenous spermidine synthase resulted in increased tolerance to these compounds without added spermidine. Increased tolerance to these fermentation inhibitors will facilitate the use of C. thermocellum, one of the most cellulolytic of all known bacterial species, for the production of fuels from plant biomass substrates.

Expression of a Cellobiose Phosphorylase from Thermotoga maritima in Caldicellulosiruptor bescii Improves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity.

Members of the genus Caldicellulosiruptor have the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species, Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass. C. bescii contains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase from Thermotoga maritima improves the phosphorolytic pathway in C. bescii and results in synergistic activity with endogenous enzymes, including CelA, to increase cellulolytic activity and growth on crystalline cellulose.IMPORTANCE CelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. This work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.

Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences.

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.

In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome.

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of Caldicellulosiruptor bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In this work, co-expression of the A. cellulolyticus Acel_0615 ß-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the ß-glucanase alone. Our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP. Biotechnol. Bioeng. 2017;114: 2474-2480. © 2017 Wiley Periodicals, Inc.

Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose.

CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C. bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain-an 82% increase over wild type CelA. In addition, expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel. Biotechnol. Bioeng. 2017;114: 945-950. © 2016 Wiley Periodicals, Inc.

Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing.

Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC) and N(4)-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.

Heterologous expression of a ß-D-glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose.

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, ß-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. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular ß-D-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted ß-D-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable ß-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 ß-D-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii.

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

Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism.

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, CO2, and H2 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.

Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725.

Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼ 80 °C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.

Detection of a novel active transposable element in Caldicellulosiruptor hydrothermalis and a new search for elements in this genus.

We show that a previously annotated hypothetical protein is the transposase of a new and active IS element, ISCahy1, widespread in Caldicellulosiruptor species. Transposition generated an 11-bp direct repeat at the insertion site in Caldicellulosiruptor hydrothermalis, suggesting a cut-and-paste mechanism. The discovery of an active insertion sequence in Caldicellulosiruptor species led to a survey of potential IS elements in the genome sequences of eight Caldicellulosiruptor species that identified several new elements, including one novel to this genus.

Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii.

Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD(680)) and more efficient DNA transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii. A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii. Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii, and may be applied broadly across the Caldicellulosiruptor genus.

Isolation and bioinformatic analysis of a novel transposable element, ISCbe4, from the hyperthermophilic bacterium, Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii is an anaerobic thermophilic bacterium of special interest for use in the consolidated bioprocessing of plant biomass to biofuels. In the course of experiments to engineer pyruvate metabolism in C. bescii, we isolated a mutant of C. bescii that contained an insertion in the L-lactate dehydrogenase gene (ldh). PCR amplification and sequencing of the ldh gene from this mutant revealed a 1,609-bp insertion that contained a single open reading frame of 479 amino acids (1,440 bp) annotated as a hypothetical protein with unknown function. The ORF is flanked by an 8-base direct repeat sequence. Bioinformatic analysis indicated that this ORF is part of a novel transposable element, ISCbe4, which is only intact in the genus Caldicellulosiruptor, but has ancient relatives that are present in degraded (and previously unrecognized) forms across many bacterial and archaeal clades.

Simultaneous expression of an endogenous spermidine synthase and a butanol dehydrogenase from Thermoanaerobacter pseudethanolicus in Clostridium thermocellum results in increased resistance to acetic acid and furans, increased ethanol production and an increase in thermotolerance.

BACKGROUND: Sensitivity to inhibitors derived from the pretreatment of plant biomass is a barrier to the consolidated bioprocessing of these complex substrates to fuels and chemicals by microbes. Spermidine is a low molecular weight aliphatic nitrogen compound ubiquitous in microorganisms, plants, and animals and is often associated with tolerance to stress. We recently showed that overexpression of the endogenous spermidine synthase enhanced tolerance of the Gram-positive bacterium, Clostridium thermocellum to the furan derivatives furfural and HMF. RESULTS: Here we show that co-expression with an NADPH-dependent heat-stable butanol dehydrogenase from Thermoanaerobacter pseudethanolicus further enhanced tolerance to furans and acetic acid and most strikingly resulted in an increase in thermotolerance at 65 °C. CONCLUSIONS: Tolerance to fermentation inhibitors will facilitate the use of plant biomass substrates by thermophiles in general and this organism in particular. The ability to grow C. thermocellum at 65 °C has profound implications for metabolic engineering.

Recombinogenic properties of Pyrococcus furiosus strain COM1 enable rapid selection of targeted mutants.

We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or "pop-out," of the selected marker. We used this method to construct a "markerless" deletion of the trpAB locus in the GLW101 (COM1 ΔpyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.