Glycoside hydrolase inventory drives plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus.

The genome of Caldicellulosiruptor saccharolyticus encodes a range of glycoside hydrolases (GHs) that mediate plant biomass deconstruction by this bacterium. Two GH-based genomic loci that appear to be central to the hydrolysis of hemicellulosic and cellulosic substrates were examined. XynB-XynF (Csac_2404-Csac_2411) encodes intracellular and extracellular GHs that are active towards xylan and xylan side-chains, as well as carboxymethyl cellulose (CMC). XynD (Csac_2409) and XynE (Csac_2410) were produced recombinantly and confirmed to be xylanases. XynF (Csac_2411) was produced in two separate polypeptides, each with one GH43 catalytic domain displaying α-L-arabinofuranosidase activity. CelA-ManB (Csac_1076-Csac_1080) encodes four multi-domain, extracellular GHs, including CelB (Csac_1078), a 118 kDa extracellular enzyme not present in the other genome-sequenced member of this genus, Caldicellulosiruptor bescii (formerly Anaerocellum thermophilum). CelB contains both GH10 and GH5 domains, separated by a family 3 carbohydrate-binding module (CBM3). CelB encoded in Csac_1078 differed from the version originally reported (Saul et al., 1990, Appl Environ Microbiol 56:3117­3124) with respect to linker regions. CelB hydrolyzed xylan and CMC, as well as barley b-glucan, glucomannan, and arabinoxylan. For all substrates tested, intact CelB was significantly more active than either the individual GH5 and GH10 domains or the two discrete domains together, indicating that the multi-domain architecture is essential for complex carbohydrate hydrolysis. Transcriptomes for C. saccharolyticus grown at 70°C on glucose, xylose, xyloglucan, switchgrass, and poplar revealed that certain GHs were particularly responsive to growth on switchgrass and poplar and that CelB was in the top decile of all transcripts during growth on the plant biomass.

Carbohydrate utilization patterns for the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus reveal broad growth substrate preferences.

Coutilization of hexoses and pentoses derived from lignocellulose is an attractive trait in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H(2)-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose, and xylose), mixtures of these sugars, as well as on xylan and xylogluco-oligosacchrides. C. saccharolyticus grew at approximately the same rate (t(d), approximately 95 min) and to the same final cell density (1 x 10(8) to 3 x 10(8) cells/ml) on all sugars and sugar mixtures tested. In the monosaccharide mixture, although simultaneous consumption of all monosaccharides was observed, not all were utilized to the same extent (fructose > xylose/arabinose > mannose/glucose/galactose). Transcriptome contrasts for monosaccharide growth revealed minimal changes in some cases (e.g., 32 open reading frames [ORFs] changed >/=2-fold for glucose versus galactose), while substantial changes occurred for cases involving mannose (e.g., 353 ORFs changed >/=2-fold for glucose versus mannose). Evidence for catabolite repression was not noted for either growth on multisugar mixtures or the corresponding transcriptomes. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette transporters and single phosphotransferase system identified in C. saccharolyticus. Although most transporter genes responded to individual monosaccharides and polysaccharides, the genes Csac_0692 to Csac_0694 were upregulated only in the monosaccharide mixture. The results presented here affirm the broad growth substrate preferences of C. saccharolyticus on carbohydrates representative of lignocellulosic biomass and suggest that this bacterium holds promise for biofuel applications.

Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus.

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO(2), and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.

Polysaccharide degradation and synthesis by extremely thermophilic anaerobes.

Extremely thermophilic fermentative anaerobes (growth T(opt) > or = 70 degrees C) have the capacity to use a variety of carbohydrates as carbon and energy sources. As such, a wide variety of glycoside hydrolases and transferases have been identified in these microorganisms. The genomes of three model extreme thermophiles-an archaeon Pyrococcus furiosus (T(opt) = 98 degrees C), and two bacteria, Thermotoga maritima (T(opt) = 80 degrees C) and Caldicellulosiruptor saccharolyticus (T(opt) = 70 degrees C)-encode numerous carbohydrate-active enzymes, many of which have been characterized biochemically in their native or recombinant forms. In addition to their voracious appetite for polysaccharide degradation, polysaccharide production has also been noted for extremely thermophilic fermentative anaerobes; T. maritima generates exopolysaccharides that aid in biofilm formation, a process that appears to be driven by intraspecies and interspecies interactions.

Functional-genomics-based identification and characterization of open reading frames encoding alpha-glucoside-processing enzymes in the hyperthermophilic archaeon Pyrococcus furiosus.

Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on alpha-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 beta-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative alpha-glucan-processing enzymes.