Many bacteria efficiently degrade lignocellulose yet the underpinning genome-wide metabolic and regulatory networks remain elusive. Here we revealed the “cellulose degradome” for the model mesophilic cellulolytic bacterium Clostridium cellulolyticum ATCC 35319, via an integrated analysis of its complete genome, its transcriptomes under glucose, xylose, cellobiose, cellulose, xylan or corn stover and its extracellular proteomes under glucose, cellobiose or cellulose.

Efficient biotechnological conversion of lignocellulosic biomass to valuable products, such as transportation biofuels, is ecologically attractive, yet requires substantially improved mechanistic understanding and optimization to become economically feasible. Cellulolytic clostridia, such as Ruminiclostridium papyrosolvens (previously Clostridium papyrosolvens), produce a wide variety of carbohydrate-active enzymes (CAZymes) including extracellular multienzyme complexes—cellulosomes with different specificities for enhanced cellulosic biomass degradation. Identification of the secretory components, especially CAZymes, during bacterial growth on lignocellulose and their influence on bacterial catalytic capabilities provide insight into construction of potent cellulase systems of cell factories tuned or optimized for the targeted substrate by matching the type and abundance of enzymes and corresponding transporters. In this study, we firstly predicted a total of 174 putative CAZymes from the genome of R. papyrosolvens, including 74 cellulosomal components. To explore profile of secreted proteins involved in lignocellulose degradation, we compared the secretomes of R. papyrosolvens grown on different substrates using label-free quantitative proteomics. CAZymes, extracellular solute-binding proteins (SBPs) of transport systems and proteins involved in spore formation were enriched in the secretome of corn stover for lignocellulose degradation. Furthermore, compared with free CAZymes, complex CAZymes (cellulosomal components) had larger fluctuations in variety and abundance of enzymes among four carbon sources. In particular, cellulosomal proteins encoded by the cip-cel operon and the xyl-doc gene cluster had the highest abundance with corn stover as substrate. Analysis of differential expression of CAZymes revealed a substrate-dependent secretion pattern of CAZymes, which was consistent with their catalytic activity from each secretome determined on different cellulosic substrates. The results suggest that the expression of CAZymes is regulated by the type of substrate in the growth medium. In the present study, our results demonstrated the complexity of the lignocellulose degradation systems of R. papyrosolvens and showed the potency of its biomass degradation activity. Differential proteomic analyses and activity assays of CAZymes secreted by R. papyrosolvens suggested a distinct environment-sensing strategy for cellulose utilization in which R. papyrosolvens modulated the composition of the CAZymes, especially cellulosome, according to the degradation state of its natural substrate.

Mesophilic, anaerobic, and cellulolytic Ruminiclostridium-type bacterial species can secrete an extracellular, multi-enzyme machinery cellulosome, which efficiently degrades cellulose. In this study, we first reported the complete genome of Ruminiclostridium papyrosolvens DSM2782, a single circular 5,027,861-bp chromosome with 37.1% G + C content, and compared it with other Ruminiclostridium-type species. Pan-genome analysis showed that Ruminiclostridium-type species share a large number of core genes to conserve basic functions, although they have a high level of intraspecific genetic diversity. Especially, KEGG mapping revealed that Ruminiclostridium-type species mainly use ABC transporters regulated by two-component systems (TCSs) to absorb extracellular sugars but not phosphotransferase systems (PTSs) that are employed by solventogenic clostridia, such as Clostridium acetobutylicum. Furthermore, we performed comparative analyses of the species-specific repertoire of CAZymes for each of the Ruminiclostridium-type species. The high similarity of their cohesins suggests a common ancestor and potential cross-species recognition. Additionally, both differences between the C-terminal cohesins and other cohesins of scaffoldins and between the dockerins linking with cellulases and other catalytic domains indicate a preference for the location of cellulosomal catalytic subunits at scaffoldins. The information gained in this study may be utilized directly or developed further by genetic engineering and optimizing enzyme systems or cell factories for enhanced biotechnological biomass deconstruction and biofuel production.

Anaerobes harbor some of the most efficient biological machinery for cellulose degradation, especially thermophilic bacteria, such as Acetivibrio thermocellus and Thermoclostridium stercorarium, which play a fundamental role in transferring lignocellulose into ethanol through consolidated bioprocessing (CBP). In this study, we compared activities of two cellulase systems under varying kinds of hemicellulose and cellulose. A. thermocellus was identified to contribute specifically to cellulose hydrolysis, whereas T. stercorarium contributes to hemicellulose hydrolysis. The two systems were assayed in various combinations to assess their synergistic effects using cellulose and corn stover as the substrates. Their maximum synergy degrees on cellulose and corn stover were, respectively, 1.26 and 1.87 at the ratio of 3:2. Furthermore, co-culture of these anaerobes on the mixture of cellulose and xylan increased ethanol concentration from 21.0 to 40.4 mM with a high cellulose/xylan-to-ethanol conversion rate of up to 20.7%, while the conversion rates of T. stercorarium and A. thermocellus monocultures were 19.3% and 15.2%. The reason is that A. thermocellus had the ability to rapidly degrade cellulose while T. stercorarium co-utilized both pentose and hexose, the metabolites of cellulose degradation, to produce ethanol. The synergistic effect of cellulase systems and metabolic pathways in A. thermocellus and T. stercorarium provides a novel strategy for the design, selection, and optimization of ethanol production from cellulosic biomass through CBP.

Anaerobic, mesophilic, and cellulolytic Clostridium papyrosolvens produces an efficient cellulolytic extracellular complex named cellulosome that hydrolyzes plant cell wall polysaccharides into simple sugars. Its genome harbors two long cellulosomal clusters: cip-cel operon encoding major cellulosome components (including scaffolding) and xyl-doc gene cluster encoding hemicellulases. Compared with works on cip-cel operon, there are much fewer studies on xyl-doc mainly due to its rare location in cellulolytic clostridia. Sequence analysis of xyl-doc revealed that it harbors a 5′ untranslated region (5′-UTR) which potentially plays a role in the regulation of downstream gene expression. Here, we analyzed the function of 5′-UTR of xyl-doc cluster in C. papyrosolvens in vivo via transformation technology developed in this study. In this study, we firstly developed an electrotransformation method for C. papyrosolvens DSM 2782 before the analysis of 5′-UTR of xyl-doc cluster. In the optimized condition, a field with an intensity of 7.5–9.0 kV/cm was applied to a cuvette (0.2 cm gap) containing a mixture of plasmid and late cell suspended in exponential phase to form a 5 ms pulse in a sucrose-containing buffer. Afterwards, the putative promoter and the 5′-UTR of xyl-doc cluster were determined by sequence alignment. It is indicated that xyl-doc possesses a long conservative 5′-UTR with a complex secondary structure encompassing at least two perfect stem-loops which are potential candidates for controlling the transcriptional termination. In the last step, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) as a quantitative reporter to analyze promoter activity and 5′-UTR function in vivo. It revealed that 5′-UTR significantly blocked transcription of downstream genes, but corn stover can relieve its suppression. In the present study, our results demonstrated that 5′-UTR of the cellulosomal xyl-doc cluster blocks the transcriptional activity of promoter. However, some substrates, such as corn stover, can relieve the effect of depression of 5′-UTR. Thus, it is speculated that 5′-UTR of xyl-doc was a putative riboswitch to regulate the expression of downstream cellulosomal genes, which is helpful to understand the complex regulation of cellulosome.