@article{zurawski_khatibi_akinosho_straub_compton_conway_lee_ragauskas_davison_adams_et al._2017, title={Bioavailability of Carbohydrate Content in Natural and Transgenic Switchgrasses for the Extreme Thermophile Caldicellulosiruptor bescii}, volume={83}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00969-17}, abstractNote={ABSTRACT Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(−) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance. IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium Caldicellulosiruptor bescii was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall. }, number={17}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Zurawski, Jeffrey V. and Khatibi, Piyum A. and Akinosho, Hannah O. and Straub, Christopher T. and Compton, Scott H. and Conway, Jonathan M. and Lee, Laura L. and Ragauskas, Arthur J. and Davison, Brian H. and Adams, Michael W. W. and et al.}, year={2017}, month={Sep} } @article{khatibi_chou_loder_zurawski_adams_kelly_2017, title={Impact of growth mode, phase, and rate on the metabolic state of the extremely thermophilic archaeon Pyrococcus furiosus}, volume={114}, ISSN={["1097-0290"]}, DOI={10.1002/bit.26408}, abstractNote={AbstractThe archaeon Pyrococcus furiosus is emerging as a metabolic engineering platform for production of fuels and chemicals, such that more must be known about this organism's characteristics in bioprocessing contexts. Its ability to grow at temperatures from 70 to greater than 100°C and thereby avoid contamination, offers the opportunity for long duration, continuous bioprocesses as an alternative to batch systems. Toward that end, we analyzed the transcriptome of P. furiosus to reveal its metabolic state during different growth modes that are relevant to bioprocessing. As cells progressed from exponential to stationary phase in batch cultures, genes involved in biosynthetic pathways important to replacing diminishing supplies of key nutrients and genes responsible for the onset of stress responses were up‐regulated. In contrast, during continuous culture, the progression to higher dilution rates down‐regulated many biosynthetic processes as nutrient supplies were increased. Most interesting was the contrast between batch exponential phase and continuous culture at comparable growth rates (∼0.4 hr−1), where over 200 genes were differentially transcribed, indicating among other things, N‐limitation in the chemostat and the onset of oxidative stress. The results here suggest that cellular processes involved in carbon and electron flux in P. furiosus were significantly impacted by growth mode, phase and rate, factors that need to be taken into account when developing successful metabolic engineering strategies.}, number={12}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Khatibi, Piyum A. and Chou, Chung-jung and Loder, Andrew J. and Zurawski, Jeffrey V. and Adams, Michael W. W. and Kelly, Robert M.}, year={2017}, month={Dec}, pages={2947–2954} } @article{conway_pierce_le_harper_wright_tucker_zurawski_lee_blumer-schuette_kelly_2016, title={Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species}, volume={291}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.707810}, abstractNote={The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis encodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptor species genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique to C. kronotskyensis and contains SLH domains. Calkro_0111 was produced recombinantly in Escherichia coli as two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo- and exoglucanase activity on the β-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs in C. kronotskyensis, when produced in E. coli, was active on a variety of xylans and β-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptor and among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii. As such, the gene encoding Calkro_0402 was inserted into the C. bescii genome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-type C. bescii. The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptor species.}, number={13}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Conway, Jonathan M. and Pierce, William S. and Le, Jaycee H. and Harper, George W. and Wright, John H. and Tucker, Allyson L. and Zurawski, Jeffrey V. and Lee, Laura L. and Blumer-Schuette, Sara E. and Kelly, Robert M.}, year={2016}, month={Mar}, pages={6732–6747} } @article{zurawski_conway_lee_simpson_izquierdo_blumer-schuette_nookaew_adams_kelly_2015, title={Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization}, volume={81}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01622-15}, abstractNote={ABSTRACT Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO 2 , and H 2 . Caldicellulosiruptor bescii , C. kronotskyensis , and C. saccharolyticus solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass ( Panicum virgatum ) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with C. saccharolyticus , not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation. }, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Zurawski, Jeffrey V. and Conway, Jonathan M. and Lee, Laura L. and Simpson, Hunter J. and Izquierdo, Javier A. and Blumer-Schuette, Sara and Nookaew, Intawat and Adams, Michael W. W. and Kelly, Robert M.}, year={2015}, month={Oct}, pages={7159–7170} } @article{blumer-schuette_alahuhta_conway_lee_zurawski_giannone_hettich_lunin_himmel_kelly_2015, title={Discrete and Structurally Unique Proteins (Tapirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose}, volume={290}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m115.641480}, abstractNote={Background: Lignocellulose-degrading microorganisms utilize binding modules associated with glycosidic enzymes to attach to polysaccharides. Results: Structurally unique, discrete proteins (tāpirins) bind to cellulose with a high affinity. Conclusion: Tāpirins represent a new class of proteins used by Caldicellulosiruptor species to attach to cellulose. Significance: The tāpirins establish a new paradigm for how cellulolytic bacteria adhere to cellulose. A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tāpirins,” origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.}, number={17}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Blumer-Schuette, Sara E. and Alahuhta, Markus and Conway, Jonathan M. and Lee, Laura L. and Zurawski, Jeffrey V. and Giannone, Richard J. and Hettich, Robert L. and Lunin, Vladimir V. and Himmel, Michael E. and Kelly, Robert M.}, year={2015}, month={Apr}, pages={10645–10656} } @misc{blumer-schuette_brown_sander_bayer_kataeva_zurawski_conway_adams_kelly_2014, title={Thermophilic lignocellulose deconstruction}, volume={38}, ISSN={["1574-6976"]}, DOI={10.1111/1574-6976.12044}, abstractNote={Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.}, number={3}, journal={FEMS MICROBIOLOGY REVIEWS}, author={Blumer-Schuette, Sara E. and Brown, Steven D. and Sander, Kyle B. and Bayer, Edward A. and Kataeva, Irina and Zurawski, Jeffrey V. and Conway, Jonathan M. and Adams, Michael W. W. and Kelly, Robert M.}, year={2014}, month={May}, pages={393–448} } @article{blumer-schuette_giannone_zurawski_ozdemir_ma_yin_xu_kataeva_poole_adams_et al._2012, title={Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass}, volume={194}, ISSN={["1098-5530"]}, DOI={10.1128/jb.00266-12}, abstractNote={ABSTRACT Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose. }, number={15}, journal={JOURNAL OF BACTERIOLOGY}, author={Blumer-Schuette, Sara E. and Giannone, Richard J. and Zurawski, Jeffrey V. and Ozdemir, Inci and Ma, Qin and Yin, Yanbin and Xu, Ying and Kataeva, Irina and Poole, Farris L., II and Adams, Michael W. W. and et al.}, year={2012}, month={Aug}, pages={4015–4028} } @article{conway_zurawski_lee_blumer-schuette_kelly, title={Lignocellulosic biomass deconstruction by the extremely thermophilic genus caldicellulosiruptor}, journal={Thermophilic Microorganisms}, author={Conway, J. M. and Zurawski, J. V. and Lee, L. L. and Blumer-Schuette, S. E. and Kelly, R. M.}, pages={91–119} }