Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus

Abstract

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.

FIG. 1.

Diagram of the C. saccharolyticus chromosome. From outside to inside the circles show (i) the genomic positions, (ii) the coding sequences on the positive negative strand, (iii) the coding sequences on the negative strand (the colors used for the coding sequences indicate the COG functional categories), (iv) the tRNA genes, (v) the G+C content, and (vi) the GC skew. The Microbial Genome Viewer was used to construct the circular chromosome wheel (21).

FIG. 2.

Overview of the carbon metabolism and transport systems in C. saccharolyticus. The identities of the various ABC-type sugar transporters are not known. Secondary transport systems may be involved as well. Abbreviations: Rhu-1-P, rhamnulose-1-phosphate; F-1-P, fructose-1-phosphate; G-1-P, glucose-1-phosphate; G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-bisP, fructose-1,6-bisphosphate; L-Ru-5-P, l-ribulose-5-phosphate; D-Ru-5-P, d-ribulose-5-phosphate; D-Xu-5-P, d-xylulose-5-phosphate; D-Ri-5-P, d-ribose-5-phosphate; DKI, 5-keto-4-deoxyuronate; DKII, 2,5-diketo-3-deoxygluconate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; KDPG, KDG phosphate; Fd^red, reduced ferredoxin; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; FADH, reduced flavin adenine dinucleotide; Ac-CoA, acetyl coenzyme A; Ac-P, acetyl phosphate; br., branched.

FIG. 3.

Growth of C. saccharolyticus on a xylose-glucose mixture (1:1, wt/wt). ○, optical density at 660 nm (OD660); ▴, hydrogen; ▵, acetate; ▪, glucose; ⧫, xylose; □, lactate.

FIG. 4.

Intensity ratios for transcript levels of selected genes that responded to growth on glucose (open bars), xylose (gray bars), or a mixture of glucose and xylose (black bars), compared to the results obtained with rhamnose. Ratios are expressed as log₂ values. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NdH, NAD-dependent hydrogenase; FdH, ferredoxin-dependent hydrogenase; dep., dependent; G6P, glucose-6-phosphate; FbisP, fructose bisphosphate; DH, dehydrogenase.