General features of the genome of C. phytofermentans.

General features of the genome of C. phytofermentans.

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Clostridium phytofermentans was isolated from forest soil and is distinguished by its capacity to directly ferment plant cell wall polysaccharides into ethanol as the primary product, suggesting that it possesses unusual catabolic pathways. The objective of the present study was to understand the molecular mechanisms of biomass conversion to ethano...

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Biomass constitutes all the plant matter found on our planet, and is produced directly by photosynthesis, the fundamental engine of life on earth. It is the photosynthetic capability of plants to utilize carbon dioxide from the atmosphere that leads to its designation as a “carbon neutral” fuel, meaning that it does not introduce new carbon into th...

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... The overabundance of Cazymes is not necessarily related to high enzymatic activity, as it depends on the genomic context such as the possible presence of a promoter (Williams-Rhaesa et al. 2018), operon (Chakraborty et al. 2021). Indeed, Clostridia phytofermentas, which is a very good lignocellulolytic degrader (Tolonen et al. 2011) and expresses several high lignocellulolytic enzymatic activities (Petit et al. 2015), secreted approximately 80 Cazymes (among the 161 present in its genome) in silico, which confirms that a very high abundance of Cazymes does not necessarily indicate excellent enzymatic activity. ...
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The Acidobacteria phylum is a very abundant group (20–30% of microbial communities in soil ecosystems); however, little is known about these microorganisms and their ability to degrade the biomass and lignocellulose due to the difficulty of culturing them. We, therefore, bioinformatically studied the content of lignocellulolytic enzymes (total and predicted secreted enzymes) and secreted peptidases in an in silico library containing 41 Acidobacteria genomes. The results showed a high abundance and diversity of total and secreted Carbohydrate-Active enzymes (cazyme) families among the Acidobacteria compared to known previous degraders. Indeed, the relative abundance of cazymes in some genomes represented more than 6% of the gene coding proteins with at least 300 cazymes. The same observation was made with the predicted secreted peptidases with several families of secreted peptidases, which represented at least 1.5% of the gene coding proteins in several genomes. These results allowed us to highlight the lignocellulolytic potential of the Acidobacteria phylum in the degradation of lignocellulosic biomass, which could explain its high abundance in the environment.
... 1−4 Clostridium phytofermentans (also called Lachnoclostridium phytofermentans) is a member of the family Lachnospiraceae that is distinguished by its ability to ferment lignocellulosic biomass with ethanol, hydrogen, and acetate as major products. 5,6 Advances in genetic manipulation are needed to exploit the therapeutic and industrial potential of additional Clostridia species. In particular, experimental approaches for in vivo modulation of gene expression have a few prerequisites. ...
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Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
... In addition to Neurospora crassa, numerous filamentous Ascomycota are known for their prolific cellulase and hemicellulase activity, which make them commonly used in industry (Shah et al., 2017). Such fungal species, including Trichoderma reesei, were detected in our protein datasets, as were a number of bacterial species capable of fermenting complex organic matter, including Clostridium phytofermentans (Petit et al., 2015) and C. cellulolyticum (Desvaux, 2005). Enterobacteriales and Saccharomycetales were also involved in hydrolysis, with Enterobacter previously reported as highly efficient hydrolysers of food waste at low pH (Yan et al., 2014). ...
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As the human population grows on the planet so does the generation of waste and particularly that of food waste. In order to tackle the world sustainability crisis, efforts to recover products from waste are critical. Here, we anaerobically recovered volatile fatty acids (VFAs) from food waste and analysed the microbial populations underpinning the process. An increased contribution of fungi relative to bacteria was observed throughout the reactor operation, with both kingdoms implicated into the main three steps of anaerobic digestion occurring within our systems: hydrolysis, acidogenesis and acetogenesis. Overall, Ascomycota, Proteobacteria and Firmicutes were found to drive the anaerobic digestion of food waste, with butyrate as the most abundant VFA likely produced by Clostridium using lactate as a precursor. Taken together we demonstrate that the generation of products of added-value from food waste results from cross-kingdoms microbial activities implicating fungi and bacteria.
... They found that 149.6 mL H 2 /g-VS yield was achieved by the addition of 37.5 mg/L of every nanoparticle. Some researchers used clostridium as a biocatalyst for different applications because clostridium species have a rich tradition in biofuel improvement [207]. Clostridium acetobutylicum has been employed for butanol, acetone and ethanol production from starch and is a profitable established valued bacterium [208]. ...
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Global population growth and accelerated urbanisation have resulted in massive amounts of fossil fuel use and waste production. Because of its high energy content, pure nature, and fuel quality, hydrogen fuel is a viable option to fossil fuels. Biohydrogen from agricultural waste, in particular, piques concern because it generates hydrogen while still disposing of waste. This review conducted a bibliometric analysis of biohydrogen production from organic waste to trace the research trends and hotspots based on the literature in the Web of Science (WOS) database from 1970 to 2020. The present review article also focuses on highlighting various processes for converting organic waste into hydrogen, raw materials for biohydrogen production, and catalysts that could distil the latest perceptions that could shed light on a route advancing for successful catalyst design. It also seems that some intentions have been paid on studying waste materials such as pure polysaccharides, disaccharides, and monosaccharides. Among all the catalysts used, non-noble and low-cost active metals over reduced graphene oxide (rGO) support can significantly affect the activity of fermentative hydrogen production from organic waste materials. However, researches focusing on developing anaerobic membrane bioreactors for these technologies are still needed.
... C. phytofermentans is remarkable among the Clostridium genus due to its ability to catabolize a broad range of substrates. Its genome encodes over 169 carbohydrate-active enzymes, the largest number among sequenced clostridia, and its efficient ethanol production makes it a model system for cellulosic biofuel production 21,23,[34][35][36][37] . E. coli is a well studied, facultative anaerobe capable of fermenting a broad range of substrates including glucose and glycerol which is a widely available waste product from biodiesel production 21,23,38 . ...
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Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans ( a.k.a. Lachnoclostridium phytofermentans ), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli , a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O 2 , creating an anoxic environment for C. phytofermentans . Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli . E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.
... L. phytofermentans is an obligately anaerobic fibrolytic bacterium that can ferment a wide range of plant polysaccharides [38]. This ability appears to be due to the numerous and diverse range of glycosyl hydrolases encoded within its genome, many of which have been acquired by horizontal gene transfer [40]. Whilst the L. phytofermentans type strain was isolated from forest soil, its optimum temperature for growth was reported to be 37°C with growth observed at pH 6.0-9.0 [38]. ...
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Background Compared to horses and ponies, donkeys have increased degradation of dietary fiber. The longer total mean retention time of feed in the donkey gut has been proposed to be the basis of this, because of the increased time available for feed to be acted upon by enzymes and the gut microbiota. However, differences in terms of microbial concentrations and/or community composition in the hindgut may also underpin the increased degradation of fiber in donkeys. Therefore, a study was conducted to assess if differences existed between the fecal microbiota of pony, donkey and hybrids derived from them (i.e. pony × donkey) when fed the same forage diet. Results Fecal community composition of prokaryotes and anaerobic fungi significantly differed between equine types. The relative abundance of two bacterial genera was significantly higher in donkey compared to both pony and pony x donkey: Lachnoclostridium 10 and ‘probable genus 10’ from the Lachnospiraceae family. The relative abundance of Piromyces was significantly lower in donkey compared to pony × donkey, with pony not significantly differing from either of the other equine types. In contrast, the uncultivated genus SK3 was only found in donkey (4 of the 8 animals). The number of anaerobic fungal OTUs was also significantly higher in donkey than in the other two equine types, with no significant differences found between pony and pony × donkey. Equine types did not significantly differ with respect to prokaryotic alpha diversity, fecal dry matter content or fecal concentrations of bacteria, archaea and anaerobic fungi. Conclusions Donkey fecal microbiota differed from that of both pony and pony × donkey. These differences related to a higher relative abundance and diversity of taxa with known, or speculated, roles in plant material degradation. These findings are consistent with the previously reported increased fiber degradation in donkeys compared to ponies, and suggest that the hindgut microbiota plays a role. This offers novel opportunities for pony and pony × donkey to extract more energy from dietary fiber via microbial mediated strategies. This could potentially decrease the need for energy dense feeds which are a risk factor for gut-mediated disease.
... T he clostridia are Gram-positive obligately anaerobic bacteria that include human pathogens as well as plant-fermenting species critical for healthy functioning of soil and gut microbiomes. Clostridium (Lachnoclostridium) phytofermentans ISDg (1) is a model plant-fermenting Clostridium that breaks down plant biomass using numerous carbohydrate-active enzymes (CAZymes) and ferments the resulting hexose and pentose sugars into ethanol, hydrogen, and acetate (2)(3)(4). C. phytofermentans is a member of the Lachnospiraceae family that is abundant in soil (5), dominates the rumen (6), and includes human gut commensals that play important roles in nutrition and intestinal health (7). Because of their ability to directly ferment lignocellulose, plant-fermenting clostridia have industrial potential for the transformation of plant biomass into valueadded chemicals. ...
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Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation. IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans . We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.
... In particular, the transport and metabolism of plant sugars by Clostridium phytofermentans (also called Lachnoclostridium phytofermentans) (1) and other bacteria that ferment lignocellulosic biomass are central to carbon flow in terrestrial and aquatic ecosystems. C. phytofermentans is an anaerobic mesophile that metabolizes plant polysaccharides, including cellulose, hemicellulose, and pectin (2)(3)(4). This bacterium expresses numerous carbohydrate-active enzymes (CAZymes) (5) to degrade plant polysaccharides into hexoses and pentoses of various chain lengths, which are taken into the cell using a panoply of transporters. ...
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Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans . We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.
... Proteins encoded by A. amylolytica were annotated using cluster of orthologous groups (COG) protein functional classification (Tatusov et al., 2003). Carbohydrate-active enzymes (CAZymes) of A. amylolytica YIM 77502 T were identified using the CAZyme Analysis Toolkit 3 (Petit et al., 2015). The glycoside hydrolase families were analyzed using HMMER software based on the Pfam database 4 (Finn et al., 2011). ...
... Of these genes, membrane transporters are membrane proteins associated with transportation of macromolecules, such as proteins, across biological membranes (Saier et al., 2006). ATP-binding cassette transporters (ABC transporters) are members of a transport system superfamily and involved in the translocation of various substrates, such as glucose, cellobiose, and galactose, across membranes (Kemner et al., 1997;Petit et al., 2015). ...
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The bioconversion of lignocellulose in various industrial processes, such as biofuel production, requires the degradation of cellulose. Actinomadura amylolytica YIM 77502T is an aerobic, Gram-positive actinomycete that can efficiently degrade crystalline cellulose by extracellular cellulases. Genomic analysis of A. amylolytica identified 9 cellulase and 11 β-glucosidase genes that could potentially encode proteins that digest cellulose. Extracellular proteome characterization of A. amylolytica cell-free culture supernatant by liquid chromatography tandem mass spectrometry analysis revealed that 4 of these cellulases and 2 of these β-glucosidases functioned during cellulose hydrolysis. Thin-layer chromatography analysis revealed extracellular β-glucosidases play a major role in carboxyl methyl cellulose (CMC) degradation of products in culture supernatants. In this study, 2 of the identified secreted β-glucosidases, AaBGL1 and AaBGL2, were functionally expressed in Escherichia coli and found to have β-glucosidase activity with wide substrate specificities, including for p-nitrophenyl β-D-glucopyranoside (pNPG), p-nitrophenyl-beta-D-cellobioside (pNPC), and cellobiose. Moreover, AaBGL1 and AaBGL2 had high tolerances for glucose. After adding these β-glucosidases to commercial cellulases, the degradation rates of CMC, Avicel, birch sawdust, and corncob powder increased by 37, 42, 33, and 9%, respectively. Overall, this work identifies an alternative potential source of β-glucosidases with potential applications in commercial cellulose utilization and the bioenergy industry.
... The sequence data described here have been deposited in JGI IMG (Submission ID: 105093) and DDBJ/ENA/GenBank (Accession number: MSZZ00000000). Carbohydrate-active enzymes (CAZymes) of T. rubra were determined using the CAZymes Analysis Toolkit (http://mothra.ornl.gov/cgi-bin/cat/cat.cgi; Petit et al., 2015). The results of GHs (Glycoside hydrolase families) were analyzed using the HMMER software based on the Pfam database (http://pfam.xfam.org/; ...
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Thermoactinospora rubra YIM 77501T is an aerobic, Gram-positive, spore-forming and cellulose degrading thermophilic actinomycete isolated from a sandy soil sample of a volcano. Its growth temperature range is 28–60°C. The genomic sequence of this strain revealed that there are 27 cellulase genes belonging to six glycoside hydrolase families. To understand the strategy that this strain uses to utilize carbon sources such as cellulose at different temperatures, comparative transcriptomics analysis of T. rubra YIM 77501T was performed by growing it with cellulose (CMC) and without cellulose (replaced with glucose) at 30, 40, and 50°C, respectively. Transcriptomic analyses showed four cellulase genes (TrBG2, TrBG3, TrBG4, and ThrCel6B) were up-regulated at 30, 40, and 50°C. The rate of gene expression of TrBG2, TrBG3, TrBG4, and ThrCel6B were 50°C > 30°C > 40°C. One cellulase gene (TrBG1) and two cellulase genes (TrBG5 and ThrCel6A) were up-regulated only at 30 and 50°C, respectively. These up-regulated cellulase genes were cloned and expressed in Escherichia coli. The enzymatic properties of up-regulated cellulases showed a variety of responses to temperature. Special up-regulated cellulases TrBG1 and ThrCel6A displayed temperature acclimation for each growth condition. These expression patterns revealed that a hybrid strategy was used by T. rubra to utilize carbon sources at different temperatures. This study provides genomic, transcriptomics, and experimental data useful for understanding how microorganisms respond to environmental changes and their application in enhancing cellulose hydrolysis for animal feed and bioenergy production.