Abstract
ABSTRACTExtremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose.IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.
Highlights
Thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides
These include six multidomain proteins containing two to five glycosyl hydrolases (GHs) domains that contain N-terminal signal peptides suggesting their extracellular localization. These secreted GHs are encoded within the glucan degradation locus (GDL), which plays an essential role in plant biomass deconstruction [6]
The secreted carbohydrate-active enzymes (CAZymes) are involved in degradation of plant-derived polymers, including cellulose, mannans, hemicelluloses, pectin, and starches, while the cytoplasmic GH enzymes have broader functionalities since they are capable of catabolizing a variety of oligo- and disaccharides to release sugars that enter the central carbohydrate metabolism of C. bescii
Summary
Thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. The imported saccharides undergo intracellular processing via sets of enzymes organized into individual catabolic pathways These yield central metabolites that are further degraded through major glycolytic pathways (glycolysis or pentose-phosphate pathway) and natively fermented to final products (e.g., acetate, lactate, or ethanol). Cellulolytic bacteria from the genus Caldicellulosiruptor possess extensive and highly diversified machinery for plant biomass degradation and carbohydrate utilization [4]. The glucan degradation locus (GDL) in C. bescii encodes six multidomain GHs/CBMs (multifunctional cellulases), three PLs (pectate lyases and rhamnogalacturonan lyase), and several other cellulose-binding proteins This arsenal of extracellular proteins participates in primary breakdown of a variety of complex polysaccharides, including cellulose, b-glucan, xylan, mannan, and pectin [6, 7]. Many metabolic gaps remain in describing CU pathways in Caldicellulosiruptor spp., and the identity of many mono- and oligosaccharide transporters is still unknown
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