Predicted extracellular glycoside hydrolases of <i>C.termitidis</i> based on PSORTb.3 analysis.

<p>Bold: GH genes with dockerin domain.</p><p>^*: Genes with SLH domain.</p

Comparative Analysis of Carbohydrate Active Enzymes in <i>Clostridium termitidis</i> CT1112 Reveals Complex Carbohydrate Degradation Ability

<div><p><i>Clostridium termitidis</i> strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, <i>Nasutitermes lujae</i>. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in <i>C. termitidis</i>, the recently sequenced strain CT1112 of <i>C. termitidis</i> was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in <i>C. termitidis</i>, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.</p></div

Modular structure of putative cohesin I domain containing proteins identified in the <i>C. termitidis</i> CT1112 genome.

<p>(a) Cter_0001; (b) Cter_0520; (c) Cter_0526; (d) Cter_3731, and, (e) Cter_0525. CBM3-carbohydrate binding module. X2- domain of unknown function which may play a role in attachment of the putative cellulosome to the cell wall. Cohesin I proteins have dockerin binding surfaces, which bind cellulosomal enzymes and are considered important in the formation of a cellulosome. Cohesins a, c and d show putative truncated ends. Cohesins b, c and e are components of a putative cellulosome related gene cluster.</p

General features of genomes of select <i>Clostridium</i> species.

<p>*Based on PSORTb 3.0 prediction and includes GHs, PLs, and CEs.</p

Comparative analysis of the number of putative CAZy sequences in selected Clostridial species.

<p>Comparative analysis of the number of putative CAZy sequences in selected Clostridial species.</p

Comparison of the number of putative dockerin containing GH sequences in selected Clostridial species.

<p><i>C.phytofermentans</i> ISDg and <i>C.stercorarium</i> DSM 8532 have no detectable dockerin domains.</p

Biomass growth measured by optical density at 600 nm and protein content for <i>C. termitidis</i> cultured on 2 g/L xylan.

<p>Data points represent the means of 3 independent replicates. Bars above and below the means represent the standard deviation.</p

Cellulosome components of <i>C. thermocellum</i>.

<p>Enzymatic components (colored differently to indicate enzyme variety) produced by anaerobic bacteria contain a dockerin domain. Dockerins bind the cohesins of a non-catalytic scaffoldin, providing a mechanism for cellulosome assembly. Scaffoldins also contain a cellulose-specific family 3 CBM (cellulose binding module) and a C-terminal dockerin domain II that targets the cellulosome to cellulose and the bacterial cell envelope, respectively.</p

Phylogenetic analysis of selected Clostridial species based on <i>cpn60</i> gene sequences.

<p>The phylogenetic tree was obtained using neighbor-joining <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104260#pone.0104260-Saitou1" target="_blank">[29]</a> provided in MEGA version 4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104260#pone.0104260-Tamura1" target="_blank">[30]</a>. Bootstrap tests with 1000 replications were conducted to examine the reliability of the interior branches. Asterisks (*) indicates the other <i>Clostridium</i> species used in CAZy comparison.</p

Genomic Evaluation of <i>Thermoanaerobacter</i> spp. for the Construction of Designer Co-Cultures to Improve Lignocellulosic Biofuel Production

<div><p>The microbial production of ethanol from lignocellulosic biomass is a multi-component process that involves biomass hydrolysis, carbohydrate transport and utilization, and finally, the production of ethanol. Strains of the genus <i>Thermoanaerobacter</i> have been studied for decades due to their innate abilities to produce comparatively high ethanol yields from hemicellulose constituent sugars. However, their inability to hydrolyze cellulose, limits their usefulness in lignocellulosic biofuel production. As such, co-culturing <i>Thermoanaerobacter</i> spp. with cellulolytic organisms is a plausible approach to improving lignocellulose conversion efficiencies and yields of biofuels. To evaluate native lignocellulosic ethanol production capacities relative to competing fermentative end-products, comparative genomic analysis of 11 sequenced <i>Thermoanaerobacter</i> strains, including a <i>de novo</i> genome, <i>Thermoanaerobacter thermohydrosulfuricus</i> WC1, was conducted. Analysis was specifically focused on the genomic potential for each strain to address all aspects of ethanol production mentioned through a consolidated bioprocessing approach. Whole genome functional annotation analysis identified three distinct clades within the genus. The genomes of Clade 1 strains encode the fewest extracellular carbohydrate active enzymes and also show the least diversity in terms of lignocellulose relevant carbohydrate utilization pathways. However, these same strains reportedly are capable of directing a higher proportion of their total carbon flux towards ethanol, rather than non-biofuel end-products, than other <i>Thermoanaerobacter</i> strains. Strains in Clade 2 show the greatest diversity in terms of lignocellulose hydrolysis and utilization, but proportionately produce more non-ethanol end-products than Clade 1 strains. Strains in Clade 3, in which <i>T. thermohydrosulfuricus</i> WC1 is included, show mid-range potential for lignocellulose hydrolysis and utilization, but also exhibit extensive divergence from both Clade 1 and Clade 2 strains in terms of cellular energetics. The potential implications regarding strain selection and suitability for industrial ethanol production through a consolidated bioprocessing co-culturing approach are examined throughout the manuscript.</p> </div