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Key Points Cellulosomes are self-assembled multienzyme complexes that are highly efficient at degrading lignocellulose, mainly owing to common substrate targeting and consequent enzyme proximity that, together, generate substrate channelling and synergistic action. Cellulosomes have been identified in several anaerobic bacteria, with each species presenting its own molecular arrangement with varying degrees of complexity. The prevalence of cellulosomes as rare but central components in various ecosystems reflects the benefits of this enzymatic strategy. The cohesin–dockerin interaction has been studied extensively and is one of the strongest non-covalent interactions known in nature. The composition of cellulosomes is regulated and varied by the nature of the growth substrate (carbon source) of the parent bacterium. The cellulosome, as one of the most efficient machineries for the degradation of plant cell walls, can potentially be used for the large-scale conversion of biomass. Owing to the modular nature of cellulosomes, cellulosomal components have been proposed for use in additional biotechnological applications, notably, together with other affinity systems. Cellulosomes are sophisticated multicomponent complexes that are used by bacteria to degrade cellulose from plant cell walls. In this review, Artzi, Bayer and Moraïs explore the structural and functional diversity of cellulosomes and their applications; for example, in microbial biofuel production. Cellulosomes are multienzyme complexes that are produced by anaerobic cellulolytic bacteria for the degradation of lignocellulosic biomass. They comprise a complex of scaffoldin, which is the structural subunit, and various enzymatic subunits. The intersubunit interactions in these multienzyme complexes are mediated by cohesin and dockerin modules. Cellulosome-producing bacteria have been isolated from a large variety of environments, which reflects their prevalence and the importance of this microbial enzymatic strategy. In a given species, cellulosomes exhibit intrinsic heterogeneity, and between species there is a broad diversity in the composition and configuration of cellulosomes. With the development of modern technologies, such as genomics and proteomics, the full protein content of cellulosomes and their expression levels can now be assessed and the regulatory mechanisms identified. Owing to their highly efficient organization and hydrolytic activity, cellulosomes hold immense potential for application in the degradation of biomass and are the focus of much effort to engineer an ideal microorganism for the conversion of lignocellulose to valuable products, such as biofuels.

Clostridium thermocellum , a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe; however, ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 Å structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum. We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin–cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as “beads on a string,” we propose that cohesin–cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.

Cells regulate metabolic fluxes to balance energy production, biosynthesis, and the efficient use of limited resources, including the finite capacity for synthesizing and maintaining metabolic enzymes. Here, we present in vivo evidence that strongly thermodynamically favorable metabolic pathways require significantly fewer enzyme resources to sustain a given flux compared to less thermodynamically favorable pathways. These findings underscore the connection between pathway thermodynamics, resource allocation, and enzyme burden, providing valuable insights for metabolic engineering strategies aimed at optimizing pathways for high flux with minimal protein cost.

In Clostridium thermocellum, there are nine RsgI factors responsible for sensing different types of substrates and regulating the transcription and expression of cellulosome genes. Within the signaling pathway of RsgI, the membrane protease RseP cleaves RsgI in its transmembrane helix, thus releasing the N-terminal fragment of RsgI from the membrane. This released RsgI N-terminal fragment is subsequently recognized and degraded by a cytoplasmic protease complex consisting of an AAA+ ATPase and ClpP protease. Previous research showed that the ClpXP complex, comprising ClpX and ClpP, is capable of recognizing and degrading the N-terminal fragment of RsgI6. However, due to the low conservation of the transmembrane helical region of RsgI, it remains unclear whether other RsgIs are similarly recognized and degraded by the same unfoldase. In this study, we employed in vitro protease assays to examine the recognition and degradation of the N-terminal fragment of each RsgI by various ClpP-unfoldase complexes. Results confirm that ClpXP is responsible for degrading the N-terminal fragments of all RsgI proteins in C. thermocellum, suggesting a degree of sequence promiscuity in substrate recognition by ClpXP. ClpXP can recognize multiple XAA sites in the transmembrane helix region of RsgI. Moreover, we unexpectedly discovered that the cytoplasmic domain influences the degradation of RsgI2-NF by ClpXP in our in vitro assay. This study provides new insights into understanding the complex regulatory mechanisms of cellulosome genes and the role of AAA+ proteases in C. thermocellum, thereby offering critical clues for unraveling the internal regulatory networks of bacteria.

Background Clostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures. Results To separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (P clo1313_2638 ::ldh* ∆ adhE ) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE , in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 °C for ethanol concentrations ≥ 10 g L −1 and was able to reach higher maximum OD 600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD 600 and relative growth rates were higher at 45 and 50 °C, compared to 55 °C, for ethanol concentrations ≥ 15 g L −1 . For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L −1 at 45 and 50 °C compared to 55 °C. Conclusion Positive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.

Clostridium ( Ruminiclostridium ) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335′s transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log 2 fold change (FC) >|1| and 106 highly DEGs (log 2 FC >|2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD , were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes ( tktAB ) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation–reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose.

An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene ( TcBG1 ) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC 50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum . High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases. Key points • Strain A9 can secrete a thermostable β-glucosidase that has high glucose tolerance • A coculture of strain A9 and C. thermocellum showed high cellulose degradation • Strain A9 achieves biological saccharification without addition of β-glucosidase

Background Clostridium thermocellum produces H 2 and ethanol, as well as CO 2 , acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase. Conclusions Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H 2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.