Carbohydrate Utilization Patterns for the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus Reveal Broad Growth Substrate Preferences

Abstract

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 , ϳ95 min) and to the same final cell density (1 ؋ 10 8 to 3 ؋ 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.Cellulose and hemicellulose comprise ϳ70% (dry basis) of plant biomass (23) and, as such, these complex carbohydrates are promising targets for alternative energy feedstocks. For biofuel production, lignocellulose must be deconstructed into fermentable sugars by various combinations of physical, physical-chemical, chemical, and biological processing steps (33). The more efficiently and economically that this is accomplished, the more attractive this material becomes for alternative fuels (25,28,37,44). For this reason, there is great interest in identifying microorganisms that can minimize or eliminate the need for physical and chemical pretreatment of plantbased biomass and are at the same time capable of simultaneously fermenting the resulting pentoses and hexoses to biofuels (17).In environmental settings, microbial survival can be based on the capacity to utilize a variety of available carbohydrates as carbon and energy sources (19,22). When complex polysaccharides are involved, as is the case for plant-based biomass, extracellular glycoside hydrolases depolymerize potential...

Absence of Diauxie during Simultaneous Utilization of Glucose and Xylose bySulfolobus acidocaldarius

“…Similar metabolisms have also been reported in Corynebacterium glutamicum (18,50) and Caldicellulosiruptor saccharolyticus (47). C. glutamicum utilized glucose and carbon sources such as gluconate (18,31) or acetate (50) simultaneously without an apparent diauxie; in other instances, glucose inhibited the metabolism of carbon sources like ethanol (4) and glutamate (29), suggesting the existence of more than one regulatory mechanism.…”

“…C. glutamicum utilized glucose and carbon sources such as gluconate (18,31) or acetate (50) simultaneously without an apparent diauxie; in other instances, glucose inhibited the metabolism of carbon sources like ethanol (4) and glutamate (29), suggesting the existence of more than one regulatory mechanism. Similarly, the anaerobic hyperthermophilic bacterium C. saccharolyticus utilized multiple sugars independent of each other and in particular different combinations of hexose and pentose sugars, with no evidence of catabolite repression (27,46,47). While the simultaneous utilization of multiple sugars is ideal for biofuel production from cellulosic biomass, the very long doubling time of this organism would seem to be an impediment to its industrial application.…”

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

“…This taxon contains cellulolytic members, such as C. obsidiansis or C. bescii (formerly Anaerocellum thermophilum). These do not possess cellulosomes, their cellulolytic system being based on secreted multifunctional Metaproteomics of cellulose methanisation F Lü et al Metaproteomics of cellulose methanisationenzymes; they usually utilise a broad range of plant materials, including crystalline cellulose, cellulose, hemicelllose, starch and pectin, with a very high hydrogen yield (van de Werken et al, 2008;VanFossen et al, 2009;Yang et al, 2009;Hamilton-Brehm et al, 2010;Lochner et al, 2011). Among the non-redundant protein groups identified for Caldicellulosiruptor, four were probably related to hemicellulose degradation (two beta-mannanases UniRef50_Q9KWY5, one acetyl xylan esterase UniRef50_F8F4V8 and one endoxylanase UniRef50_ E4Q5G9).…”

Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass

“…Overall, the genus Caldicellulosiruptor has 6 core ATP-binding cassette (ABC) transporters out of 45 in the pangenome (see Table S4 in the supplemental material). Substrate preferences for five of these core transporters have previously been assigned based on transcriptomic analysis of C. saccharolyticus (91). Only C. hydrothermalis, C. kronotskyensis, and C. saccharolyticus contain unique transporters not found in the other sequenced Caldicellulosiruptor species.…”

“…Functional classification of proteins was determined based on searches against databases from NCBI (COG) (83), CAZy (13), integrated microbial genomes (IMG) (53), and InterProScan sequence search (100). Predictions of carbohydrate transporters were done as previously described (91) and also utilized the Find Functions database of the IMG portal (53).…”