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The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility., Competing Interests: Declarations. Ethics approval and consent to participate: This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

Cellulose from lignocellulosic biomass (LB) is of increasing interest for the production of commodity chemicals. However, its use as substrate for fermentations is a challenge due to its structural complexity. In this context, the highly cellulolytic Clostridium cellulovorans has been considered an interesting microorganism for the breakdown of LB. C. cellulovorans does not naturally produce solvents in useful concentrations, but this could be achieved by metabolic engineering. Unfortunately, this is hampered by the lack of tools for genetic engineering. We describe a genetic system that allows strain engineering by the allelic-coupled exchange method. First, the Gram-positive origin of pUB110 was identified as a suitable clostridial 'pseudo-suicide' origin of replication for the construction of deletion vectors. Second, an efficient counterselection strategy based on a codBA cassette and the use of 5-fluorocytosine as the counterselective compound was employed. Third, since the prevention of DNA transfer by host restriction-modification (RM) systems is a critical barrier to genome engineering, deletion plasmids containing flanking regions for the putative type I (Clocel_1114) and III (Clocel_2651) RM systems were constructed and transferred into C. cellulovorans. The restriction-less strains C. cellulovorans ΔClocel_1114 and C. cellulovorans ΔClocel_2651 exhibit high conjugation efficiency and can be easily used for further metabolic engineering., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflicts of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency., (© 2023. The Author(s).)

Methane (CH4) has attracted attention as not only one of the hydrogen carriers in terms of energy density, but also synthetic natural gas. In nature, the decomposition of organic compounds is performed with bacterial ecosystems that can produce CH4. Clostridium cellulovorans as a decomposer was cultivated with pig manure (PM) as an unused biomass in this study. As a result of high-performance liquid chromatography (HPLC) analysis, while formate and lactate were decreased in the C. cellulovorans medium containing 0.5% PM, acetate and butyrate were increased in it. Accordingly, in order to compare with the effect of carbon sources for methane production, the cocultivation of C. cellulovorans and the methanogenesis of Methanosarcina mazei or microbial flora of methane production (MFMP) was carried out in the C. cellulovorans medium. As a result, only the cocultivation with C. cellulovorans and MFMP showed methane production in 0.5% acetate medium. Moreover, in comparison with a carbon source in either 1% acetate or 1% methanol medium, MFMP was only cultivated after being precultivated with 0.5% glucose medium for 12 h. The results revealed that MFMP with a 1% methanol medium produced methane approximately eight times higher than with 1% acetate medium. After cultivation with 1% acetate or 1% methanol, next-generation sequencing (NGS) analysis of MFMP was carried out. Interestingly, Methanofollis (0.211%), belonging to methanogens through the CO2 reduction pathway, was dominant in the 1% acetate medium for 72 h cultivation, while Methanosarcina siciliae (1.178%), M. barkeri (0.571%), and Methanofollis (0.490%) were major species in 1% methanol medium for 72 h cultivation. Since Methanosarcina spp. belong to acetoclasts (acetoclastic pathway), methanol could promote the growth of Methanosarcina spp., rather than acetate. Therefore, it seems that Methanosarcina spp. may play a key methanogenesis role in MFMP. Thus, these results will provide important information for low-cost biomethane production.

The degradation of recalcitrant polysaccharides such as cellulose and chitin requires the synergistic functionality of processive glycosidase (GH) cocktails. Understanding the fundamental phenomenon of processivity is of biological and economic importance for the conversion of biomass into biofuel. In this work, cellulase family 9 from Clostridium cellulovorans (Cel9G), which is a processive endoglucanase, was used to elucidate the processive binding mechanism with respect to polysaccharides, since it exhibits a multimodular crystallographic structure. Metadynamics and molecular dynamics simulations were performed to explore the dynamics of cellulose chain binding to Cel9G via processive motion. The processive movement of the cellulose chain towards the catalytic domain may exhibit several local minima, which are related to strong CH/π interactions between the sugar rings and the aromatic residues distributed at the active site. For the binding of the G6 and G12 molecules, the energy barriers were determined to be 4.8 and 7.4 kcal mol -1 , respectively. Based on the site-directed mutagenesis simulations of Y520A, it was found that the existence of Y520 is critical for processive binding. It is likely that Y520 and H125/Y416 form two anchor points to facilitate processive binding to polysaccharides. More importantly, the straight-line morphology of the substrate could be observed after the formation of the so-called slide mode, which is different from the V-shaped Michaelis complex structure revealed by quantum mechanics/molecular mechanics simulations. This indicates that an additional step, namely, catalytic activation, probably exists between processive binding and the hydrolysis reaction. Finally, a four-step catalytic cycle was proposed for Cel9G. Our work provides novel molecular-level insights into the structure-function relationship for the processive enzyme Cel9G and should aid the development of improved GH cocktails for the efficient cleavage of glycosidic linkages.

Carbohydrate degradation catalyzed by glucoside hydrolases (GHs) is a major mechanism in biomass conversion. GH family 9 endoglucanase (Cel9G) from Clostridium cellulovorans , a typical multimodular enzyme, contains a catalytic domain closely linked to a family 3c carbohydrate-binding module (CBM3c). Unlike the conventional behavior proposed for other carbohydrate-binding modules, CBM3c has a direct impact on catalytic activity. In this work, extensive molecular dynamics (MD) simulations were employed to clarify the functional role of CBM3c. Furthermore, the detailed catalytic mechanism of Cel9G was investigated at the atomistic level using the combined quantum mechanical and molecular mechanical (QM/MM) method. Based on these simulations, owing to the rigidity of the peptide linker, CBM3c may affect the enzymatic activity via direct interactions with alpha helix 4 of GH9, especially with the K123 and H125 residues. In addition, using cellohexaose as a substrate, the QM/MM MD simulations confirmed that this enzyme can cleave the β-1,4-glycosidic linkage via an inverting mechanism. An oxocarbenium ion-like transition state was located with a barrier height of 19.6 kcal mol -1 . Furthermore, the G(-1) pyranose unit preferentially adopted a distorted 1 S 5 / 4 H 5 conformer in the enzyme-substrate complex. For the cleavage of the glycosidic bond, we were able to identify a plausible route ( 1 S 5 / 4 H 5 → [ 4 H 5 / 4 E] # → 4 C 1 ) from the reactant to the product at the G(-1) site.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA CA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P) + oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr CA -thlA CA -hbd CT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing., (© 2021 Wiley Periodicals LLC.)

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2 nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis., (© 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

The goal of this work was the autodisplay of the endo β-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and β-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants V max and K m were 149 U/g DCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu 2+ , Zn 2+ and Hg 2+ as well as EDTA, detergents, and organic acids, and improved by Ca 2+ , Co 2+ and Mn 2+ ions. Ca 2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl 2 were added. Also, Ca 2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Clostridium cellulovorans is among the most promising candidates for consolidated bioprocessing (CBP) of cellulosic biomass to liquid biofuels (ethanol, butanol). C. cellulovorans metabolizes all the main plant polysaccharides and mainly produces butyrate. Since most butyrate and butanol biosynthetic reactions from acetyl-CoA are common, introduction of single heterologous alcohol/aldehyde dehydrogenase can divert the branching-point intermediate (butyryl-CoA) towards butanol production in this strain. However, engineering C. cellulovorans metabolic pathways towards industrial utilization requires better understanding of its metabolism. The present study aimed at improving comprehension of cellulose metabolism in C. cellulovorans by comparing growth kinetics, substrate consumption/product accumulation and whole-cell soluble proteome (data available via ProteomeXchange, identifier PXD015487) with those of the same strain grown on a soluble carbohydrate, glucose, as the main carbon source. Growth substrate-dependent modulations of the central metabolism were detected, including regulation of several glycolytic enzymes, fermentation pathways (e.g. hydrogenase, pyruvate formate lyase, phosphate transacetylase) and nitrogen assimilation (e.g. glutamate dehydrogenase). Overexpression of hydrogenase and increased ethanol production by glucose-grown bacteria suggest a more reduced redox state. Higher energy expenditure seems to occur in cellulose-grown C. cellulovorans (likely related to overexpression and secretion of (hemi-)cellulases), which induces up-regulation of ATP synthetic pathways, e.g. acetate production and ATP synthase. SIGNIFICANCE: C. cellulovorans can metabolize all the main plant polysaccharides (cellulose, hemicelluloses and pectins) and, unlike other well established cellulolytic microorganisms, can produce butyrate. C. cellulovorans is therefore among the most attractive candidates for direct fermentation of lignocellulose to high-value chemicals and, especially, n-butanol, i.e. one of the most promising liquid biofuels for the future. Recent studies aimed at engineering n-butanol production in C. cellulovorans represent milestones towards production of biofuels through one-step fermentation of lignocellulose but also indicated that more detailed understanding of the C. cellulovorans central carbon metabolism is essential to refine metabolic engineering strategies towards improved n-butanol production in this strain. The present study helped identifying key genes associated with specific catabolic reactions and indicated modulations of central carbon metabolism (including redox and energy balance) associated with cellulose consumption. This information will be useful to determine key enzymes and possible metabolic bottlenecks to be addressed towards improved metabolic engineering of this strain., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The cellulolytic system of Clostridium cellulovorans mainly consisting of a cellulosome that synergistically collaborates with non-complexed enzymes was investigated using cellulosic biomass. The cellulosomes were isolated from the culture supernatants with shredded paper, rice straw and sugarcane bagasse using crystalline cellulose. Enzyme solutions, including the cellulosome fractions, were analyzed by SDS-PAGE and Western blot using an anti-CbpA antibody. As a result, C. cellulovorans was able to completely degrade shredded paper for 9 days and to be continuously cultivated by the addition of new culture medium containing shredded paper, indicating, through TLC analysis, that its degradative products were glucose and cellobiose. Regarding the rice straw and sugarcane bagasse, while the degradative activity of rice straw was most active using the cellulosome in the culture supernatant of rice straw medium, that of sugarcane bagasse was most active using the cellulosome from the supernatant of cellobiose medium. Based on these results, no alcohols were found when C. acetobutylicum was cultivated in the absence of C. cellulovorans as it cannot degrade the cellulose. While 1.5 mM of ethanol was produced with C. cellulovorans cultivation, both n-butanol (1.67 mM) and ethanol (1.89 mM) were detected with the cocultivation of C. cellulovorans and C. acetobutylicum. Regarding the enzymatic activity evaluation against rice straw and sugarcane bagasse, the rice straw cellulosome fraction was the most active when compared against rice straw. Furthermore, since we attempted to choose reaction conditions more efficiently for the degradation of sugarcane bagasse, a wet jet milling device together with L-cysteine as a reducing agent was used. As a result, we found that the degradation activity was almost twice as high with 10 mM L-cysteine compared with without it. These results will provide new insights for biomass utilization.

Methane (CH4) has attracted attention as not only one of the hydrogen carriers in terms of energy density, but also synthetic natural gas. In nature, the decomposition of organic compounds is performed with bacterial ecosystems that can produce CH4. Clostridium cellulovorans as a decomposer was cultivated with pig manure (PM) as an unused biomass in this study. As a result of high-performance liquid chromatography (HPLC) analysis, while formate and lactate were decreased in the C. cellulovorans medium containing 0.5% PM, acetate and butyrate were increased in it. Accordingly, in order to compare with the effect of carbon sources for methane production, the cocultivation of C. cellulovorans and the methanogenesis of Methanosarcina mazei or microbial flora of methane production (MFMP) was carried out in the C. cellulovorans medium. As a result, only the cocultivation with C. cellulovorans and MFMP showed methane production in 0.5% acetate medium. Moreover, in comparison with a carbon source in either 1% acetate or 1% methanol medium, MFMP was only cultivated after being precultivated with 0.5% glucose medium for 12 h. The results revealed that MFMP with a 1% methanol medium produced methane approximately eight times higher than with 1% acetate medium. After cultivation with 1% acetate or 1% methanol, next-generation sequencing (NGS) analysis of MFMP was carried out. Interestingly, Methanofollis (0.211%), belonging to methanogens through the CO2 reduction pathway, was dominant in the 1% acetate medium for 72 h cultivation, while Methanosarcina siciliae (1.178%), M. barkeri (0.571%), and Methanofollis (0.490%) were major species in 1% methanol medium for 72 h cultivation. Since Methanosarcina spp. belong to acetoclasts (acetoclastic pathway), methanol could promote the growth of Methanosarcina spp., rather than acetate. Therefore, it seems that Methanosarcina spp. may play a key methanogenesis role in MFMP. Thus, these results will provide important information for low-cost biomethane production. [ABSTRACT FROM AUTHOR]

Abstract Background Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. Results We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8–19 cellulosomal proteins and 9–16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. Conclusion In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

With high cellulolytic and acetic/butyric acids production abilities, Clostridium cellulovorans is promising for use to produce cellulosic n-butanol. Here, we introduced three different aldehyde/alcohol dehydrogenases encoded by bdhB, adhE1, and adhE2 from Clostridium acetobutylicum into C. cellulovorans and studied their effects on ethanol and n-butanol production. Compared to AdhE2, AdhE1 was more specific for n-butanol biosynthesis over ethanol. Co-expressing adhE1 with bdhB produced a comparable amount of butanol but significantly less ethanol, leading to a high butanol/ethanol ratio of 7.0 and 5.6 (g/g) in glucose and cellulose fermentation, respectively. Co-expressing adhE1 or adhE2 with bdhB did not increase butanol production because the activity of BdhB was limited by the NADPH availability in C. cellulovorans. Overall, the strain overexpressing adhE2 alone produced the most n-butanol (4.0 g/L, yield: 0.22 ± 0.01 g/g). Based on the insights from this study, further metabolic engineering of C. cellulovorans for cellulosic n-butanol production is suggested., (Copyright © 2019. Published by Elsevier Ltd.)

Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n -butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n -butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n -butanol tolerance of C. cellulovorans to fully utilize its n -butanol output potential. To further improve n -butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n -butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n -butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n -butanol fermentation from lignocellulose. IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n -butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n -butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n -butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n -butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n -butanol production from lignocellulose by CBP., (Copyright © 2019 American Society for Microbiology.)

Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole-genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901–902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate-degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non-cellulosomal genes encoding hemicellulases and pectin-degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.

As an anaerobic butyrate-producing bacterium, Clostridium cellulovorans can secrete a variety of extracellular enzymes to degrade plant-based cellulose. However, with glucose as the carbon source, it still secretes a large amount of protein in the broth. The metabolism and regulation are obscure and need to be further studied. Hence, in this study, C. cellulovorans was used to conduct fed-batch fermentation of glucose and microcrystalline at pH 7.0 to produce a higher level of butyrate in the bioreactor. It produced 16.8 mM lactate, 22.3 mM acetate, and 132.7 mM butyrate in 72 h during glucose fermentation. In contrast, it produced only 11.5 mM acetate and 93.9 mM butyrate and took 192 h to complete the fermentation with cellulose as the carbon source. Furthermore, there was no lactate detected in the broth. The analysis of carbon source balance and redox balance showed that 57% of the glucose was consumed to form acids in glucose fermentation, while only 47% of the cellulose was used for acid generation in the cellulose fermentation. Meanwhile, a large amount of protein was detected in the fermentation broth in both glucose (0.9 ± 0.1 g/L) and cellulose (1.1 ± 0.2 g/L) fermentation. These results showed that protein was also a main product. C. cellulovorans metabolized glucose to generate intermediate metabolites and reducing powers (NADH and Fdred), then protein and acid synthesis consumed this reducing power to maintain the carbon source balance and redox balance in the cell metabolism. The results of comparative transcriptomics and comparative proteomics also supported the above conclusion. The method of studying the protein during Clostridium species fermentation provides a new perspective for further study on metabolic regulation.

Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 μM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

This study demonstrates that the consortium, which consists of the microbial flora of methane production (MFMP) and Clostridium cellulovorans grown with cellulose, can perform the direct conversion of cellulosic biomass to methane. The MFMP was taken from a commercial methane fermentation tank and was extremely complicated. Therefore, C. cellulovorans grown with cellobiose could not perform high degradation ability on cellulosic biomass due to competition by various microorganisms in MFMP. Focusing on the fact that C. cellulovorans was cultivated with cellulose, which is armed with cellulosome, so that it is now armed C. cellulovorans; the direct conversion was carried out by the consortium which consisted of MFMP and the armed C. cellulovorans. As a result, the consortium of C. cellulovorans grown with cellobiose and MFMP (CCeM) could not degrade the purified cellulose and mandarin orange peel. However, MFMP and the armed C. cellulovorans reduced 78.4% of the total sugar of the purified cellulose such as MN301, and produced 6.89 mL of methane simultaneously. Furthermore, the consortium consisted of MFMP and the armed C. cellulovorans degraded mandarin orange peel without any pretreatments and produced methane that was accounting for 66.2% of the total produced gas.

Abstract This study was demonstrated with a coculture fermentation system using sugar beet pulp (SBP) as a carbon source combining the cellulose-degrading bacterium Clostridium cellulovorans with microbial flora of methane production (MFMP) for the direct conversion of cellulosic biomass to methane (CH4). The MFMP was taken from a commercial methane fermentation plant and extremely complicated. Therefore, the MFMP was analyzed by a next-generation sequencing system and the microbiome was identified and classified based on several computer programs. As a result, Methanosarcina mazei (1.34% of total counts) and the other methanogens were found in the MFMP. Interestingly, the simultaneous utilization of hydrogen (H2) and carbon dioxide (CO2) for methanogenesis was observed in the coculture with Consortium of C. cellulovorans with the MFMP (CCeM) including M. mazei. Furthermore, the CCeM degraded 87.3% of SBP without any pretreatment and produced 34.0 L of CH4 per 1 kg of dry weight of SBP. Thus, a gas metabolic shift in the fermentation pattern of C. cellulovorans was observed in the CCeM coculture. These results indicated that degradation of agricultural wastes was able to be carried out simultaneously with CH4 production by C. cellulovorans and the MFMP.

Abstract For a resolution of reducing carbon dioxide emission and increasing food production to respond to the growth of global population, production of biofuels from non-edible biomass is urgently required. Abundant orange wastes, such as peel and strained lees, are produced as by-product of orange juice, which is available non-edible biomass. However, d-limonene included in citrus fruits often inhibits yeast growth and makes the ethanol fermentation difficult. This study demonstrated that isopropanol-butanol-ethanol fermentation ability of Clostridium beijerinckii and cellulosic biomass degrading ability of C. cellulovorans were cultivated under several concentrations of limonene. As a result, C. cellulovorans was able to grow even in the medium containing 0.05% limonene (v/v) and degraded 85% of total sugar from mandarin peel and strained lees without any pretreatments. More interestingly, C. beijerinckii produced 0.046 g butanol per 1 g of dried strained lees in the culture supernatant together with C. cellulovorans.

The cellulolytic system of Clostridium cellulovorans mainly consisting of a cellulosome that synergistically collaborates with non-complexed enzymes was investigated using cellulosic biomass. The cellulosomes were isolated from the culture supernatants with shredded paper, rice straw and sugarcane bagasse using crystalline cellulose. Enzyme solutions, including the cellulosome fractions, were analyzed by SDS-PAGE and Western blot using an anti-CbpA antibody. As a result, C. cellulovorans was able to completely degrade shredded paper for 9 days and to be continuously cultivated by the addition of new culture medium containing shredded paper, indicating, through TLC analysis, that its degradative products were glucose and cellobiose. Regarding the rice straw and sugarcane bagasse, while the degradative activity of rice straw was most active using the cellulosome in the culture supernatant of rice straw medium, that of sugarcane bagasse was most active using the cellulosome from the supernatant of cellobiose medium. Based on these results, no alcohols were found when C. acetobutylicum was cultivated in the absence of C. cellulovorans as it cannot degrade the cellulose. While 1.5 mM of ethanol was produced with C. cellulovorans cultivation, both n-butanol (1.67 mM) and ethanol (1.89 mM) were detected with the cocultivation of C. cellulovorans and C. acetobutylicum. Regarding the enzymatic activity evaluation against rice straw and sugarcane bagasse, the rice straw cellulosome fraction was the most active when compared against rice straw. Furthermore, since we attempted to choose reaction conditions more efficiently for the degradation of sugarcane bagasse, a wet jet milling device together with L-cysteine as a reducing agent was used. As a result, we found that the degradation activity was almost twice as high with 10 mM L-cysteine compared with without it. These results will provide new insights for biomass utilization. [ABSTRACT FROM AUTHOR]

Clostridium cellulovorans DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, C. cellulovorans was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene ( ter ), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating xylR ( Clocel_0594 ) and araR ( Clocel_1253 ), as well as overexpressing xylT ( CA_C1345 ), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of n -butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing.

In the production of biofuel from lignocellulose biomass, particularly in the case of consolidated bioprocessing where the saccharification and fermentation steps take place within the same bioreactor, many compounds may be present that could affect the enzymes within such a bioreactor. This study examined the effect of ethanol, butanol, propanol, lignin, -coumaric acid, and gallic acid on the activity of XynA from C. cellulovorans. XynA from C. cellulovorans was purified, and the effects of various compounds on enzyme activity were assayed using the dinitrosalicylic acid method. In this study, it was found that XynA was very tolerant to ethanol and only lost 25% of activity even at high concentrations of ethanol. In the presence of lignin, XynA was inhibited at very low levels and retained ~85% of its activity. The highest degree of inhibition of XynA was experienced in the presence of -coumaric acid (38%) and gallic acid (47%). The results indicate that the most problematic compounds within the bioreactor are likely to be soluble lignin degradation products resulting from pretreatment steps. Therefore, the removal of these compounds prior to saccharification should result in increased productivity within a bioreactor. This study indicates that XynA may be a suitable hemicellulase for use in bioethanol production, as it has very high tolerance for ethanol inhibition.

As an anaerobic butyrate-producing bacterium, Clostridium cellulovorans can secrete a variety of extracellular enzymes to degrade plant-based cellulose. However, with glucose as the carbon source, it still secretes a large amount of protein in the broth. The metabolism and regulation are obscure and need to be further studied. Hence, in this study, C. cellulovorans was used to conduct fed-batch fermentation of glucose and microcrystalline at pH 7.0 to produce a higher level of butyrate in the bioreactor. It produced 16.8 mM lactate, 22.3 mM acetate, and 132.7 mM butyrate in 72 h during glucose fermentation. In contrast, it produced only 11.5 mM acetate and 93.9 mM butyrate and took 192 h to complete the fermentation with cellulose as the carbon source. Furthermore, there was no lactate detected in the broth. The analysis of carbon source balance and redox balance showed that 57% of the glucose was consumed to form acids in glucose fermentation, while only 47% of the cellulose was used for acid generation in the cellulose fermentation. Meanwhile, a large amount of protein was detected in the fermentation broth in both glucose (0.9 ± 0.1 g/L) and cellulose (1.1 ± 0.2 g/L) fermentation. These results showed that protein was also a main product. C. cellulovorans metabolized glucose to generate intermediate metabolites and reducing powers (NADH and Fdred), then protein and acid synthesis consumed this reducing power to maintain the carbon source balance and redox balance in the cell metabolism. The results of comparative transcriptomics and comparative proteomics also supported the above conclusion. The method of studying the protein during Clostridium species fermentation provides a new perspective for further study on metabolic regulation. [ABSTRACT FROM AUTHOR]

Background: Butanol is an industrial commodity and also considered to be a more promising gasoline substitute compared to ethanol. Renewed attention has been paid to solvents (acetone, butanol and ethanol) production from the renewable and inexpensive substrates, for example, lignocellulose, on account of the depletion of oil resources, increasing gasoline prices and deteriorating environment. Limited to current tools for genetic manipulation, it is difficult to develop a genetically engineered microorganism with combined ability of lignocellulose utilization and solvents production. Mixed culture of cellulolytic microorganisms and solventogenic bacteria provides a more convenient and feasible approach for ABE fermentation due to the potential for synergistic utilization of the metabolic pathways of two organisms. But few bacteria pairs succeeded in producing biobutanol of high titer or high productivity without adding butyrate. The aim of this work was to use Clostridium cellulovorans 743B to saccharify lignocellulose and produce butyric acid, instead of adding cellulase and butyric acid to the medium, so that the soluble sugars and butyric acid generated can be subsequently utilized by Clostridium beijerinckii NCIMB 8052 to produce butanol in one pot reaction., Results: A stable artificial symbiotic system was constructed by co-culturing a celluloytic, anaerobic, butyrate-producing mesophile (C. cellulovorans 743B) and a non-celluloytic, solventogenic bacterium (C. beijerinckii NCIMB 8052) to produce solvents by consolidated bioprocessing (CBP) with alkali extracted deshelled corn cobs (AECC), a low-cost renewable feedstock, as the sole carbon source. Under optimized conditions, the co-culture degraded 68.6 g/L AECC and produced 11.8 g/L solvents (2.64 g/L acetone, 8.30 g/L butanol and 0.87 g/L ethanol) in less than 80 h. Besides, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the dynamics of the abundance of each strain during the co-culturing process, which figured out the roles of each strain at different periods in the symbiosis., Conclusion: Our work illustrated the great potential of artificial symbiosis in biofuel production from lignocellulosic biomass by CBP. The dynamics of the abundance of C. beijerinckii and C. cellulovorans revealed mechanisms of cooperation and competition between the two strains during the co-culture process.

The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.