Functional Genomic, Microbiological and Biochemical Characterization of Plant Biomass Deconstruction by the Extrememly Thermophilic Bacterium Caldicellulosiruptor saccharolyticus.

摘要

Rising energy prices, depletion of petroleum reserves and global warming have made the development of renewable fuels a national priority. Molecular hydrogen as a transportation fuel looms as an ethanol alternative, since it produces only water as a by-product. A promising route to hydrogen is microbial because it is less environmentally intensive than traditional chemical or electrochemical processes. Caldicellosiruptor saccharolyticus (Csac) , an extremely thermophilic bacterium, produces hydrogen from cellulose-, hemicellulose and pectin-containing plant biomass. To understand the growth physiology of this microorganism with an eye towards optimizing bioenergy yields, the Csac genome was sequenced and a whole genome oligonucleotide microarray was developed and implemented. Csac was studied with focus on plant biomass deconstruction. Previous studies indicated that this bacterium co-fermented glucose and xylose without carbon catabolite repression (CCR), an attractive physiological characteristic since these two sugars are the major components of lignocellulose hydrolysates. Here, growth on glucose, xylose, galactose, fructose, mannose and arabinose, and a mixture of all six monosaccharides indicated that Csac preferred fructose (a hexose) and xylose and arabinose (both pentoses). No microbiological or transcriptomic evidence for CCR was found during growth on monosaccharide mixtures. Transcriptomes from growth on monosaccharides and hemicelluloses were used to predict sugar substrates for most of the 24 putative carbohydrate ATP-binding cassette (ABC) transporters and one phosphotransferase system (PTS) identified in the Csac genome. Broad growth substrate preferences, diversity of ABC sugar transporters, and concomitant lack of CCR support Csac's capacity to deconstruct plant biomass.;Another distinct feature of Csac and other similar organisms is the presence of novel multi-domain glycoside hydrolases. This can also be attributed to two glycoside hydrolase (GH)-laden loci in the Csac genome (Csac_1076-Csac_1080 and Csac_2404-2411). Csac_2404-Csac_2411 encodes intracellular and extracellular GH10 glycoside hydrolases that were found to not only breakdown xylan and xylan side-chains, but also hydrolyze carboxymethyl cellulose (CMC). Csac_1076-Csac_1080 encodes several multi-domain, extracellular glycoside hydrolases (GH). Within this locus is CelB (Csac_1078), a 118 kDa enzyme so far unique to C. saccharolyticus, constructed of both a GH10 and GH5 domain separated by a family 3 carbohydrate-binding module (CBM). CelB hydrolyzed xylan and CMC, as well as barley beta-glucan, glucomannan and arabinoxylan. CelB transcripts were among the most highly transcribed (top 10%) in C. saccharolyticus during growth on switchgrass and poplar.;Given the physiological and biochemical characteristics of Csac , this bacterium has the potential for consolidated bioprocessing (biomass deconstruction and conversion to H2 by a single microorganism) and merits additional study in expanding efforts to replace petroleum with biofuels produced from renewable feedstocks.