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Summary Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism the...
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... filter paper in vivo. Cphy3367 may have the central role in cellulose degradation, with other cellu- lases having accessory functions. Alternatively, some or all of the C. phytofermentans cellulases may have required non-redundant functions to break down cellu- lose such that inactivation of these genes would have similar phenotypes to AT02 (Fig. 7A). Future gene inac- tivation studies will reveal the relative contributions of other C. phytofermentans hydrolases to cellulose ...
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... and Reilly, 2005) where the CBM is a wedge to pry cellulose chains from the crystalline cellulose surface. The CBM then functions as a Brownian ratchet to pass the freed chain to the GH domain for cleavage. Similarly, C. phytofermentans may require Cphy3367 to free cellulose chains from the cellulose fibril for attack by other cellulases (Fig. ...
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... and the cell wall ( Kosugi et al., 2004), and have thus been proposed both to promote cellulose deg- radation and to anchor the cellulosome to the cell surface. Similarly, the DUF291 domains of Cphy3367 may be criti- cal to degrade cellulose or to attach Cphy3367 to the cell surface while the CBM domains bind Cphy3367 to the cellulosic substrate (Fig. 7C). The inability of AT02 to degrade cellulose could be due, at least in part, to an inability to retain the cell in close enough proximity to the cellulose substrate so that hydrolases can function ...
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Citations
... The second method is based on the Ll.LtrB mobile group II intron derived from Lactococcus lactis, named ClosTron (Heap et al. 2007), in which the intron-encoded protein (IEP) can reverse transcribe intron RNA into cDNA and assists the cDNA in fully inserting into the target of genome through the DNA recombination and repair mechanism in the cell that leads to gene disruption (Karberg et al. 2001;Lambowitz and Zimmerly 2011). It has been known to be very effective in clostridia (Cai et al. 2011;Chen et al. 2005;Heap et al. 2010;Kuehne et al. 2010;Li et al. 2012;Mohr et al. 2013;Shao et al. 2007;Tolonen et al. 2009). Recently, genome editing based on various CRISPR-Cas systems has been developed in several clostridia. ...
Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its metabolic engineering is limited by lack of genetic tools. Here, we firstly employed the endogenous xylan-inducible promoter to control ClosTron system for gene disruption of R. papyrosolvens. The modified ClosTron can be easily transformed into R. papyrosolvens and specifically disrupt targeting genes. Furthermore, a counter selectable system based on uracil phosphoribosyl-transferase (Upp) was successfully established and introduced into the ClosTron system, which resulted in plasmid curing rapidly. Thus, the combination of xylan-inducible ClosTron and upp-based counter selectable system makes the gene disruption more efficient and convenient for successive gene disruption in R. papyrosolvens.
Key points
• Limiting expression of LtrA enhanced the transformation of ClosTron plasmids in R. papyrosolvens.
• DNA targeting specificity can be improved by precise management of the expression of LtrA.
• Curing of ClosTron plasmids was achieved by introducing the upp-based counter selectable system.
... Targeted gene inactivation (TGI) is a proper technique for the study of a gene: its function, its role in a cell's life, its association with specific traits, its ability to cause disease, etc. (Meng et al. 2008;Tolonen et al. 2009;Zhang et al. 2019;Wang et al. 2022). Also, TGI is a major goal in the treatment of specific diseases like cancer (Setton et al. 2021;Duffy 2020). ...
Background:
Targeted gene inactivation (TGI) is a widely used technique for the study of genes' functions. There are many different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineering tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed repair (HDR).
Methods:
For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombination was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme. pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed bacteria were screened for recombination using PCR, restriction digestion and sequencing.
Results:
The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which the gidA gene is disrupted.
Conclusion:
In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria's genes.
... This ratio is significantly higher than that of the whole gene set (291 of 7423 genes encode secreted proteins). The purple module includes homologous genes of Axe1 (regulating fungal pathogenicity), xylanase (xylan degradation), and glucanases (cellulose degradation), which are very important for regulating pathogenicity [41][42][43]. It likely represents a specific virulence module. ...
Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study.
... Therefore, targeted inactivation of a gene at locus Cphy3367 in Clostridium phytofermentans illustrates that degradation of cellulosic substrate requires the GH9 enzymes, as the gene is known to encode major Cel9A enzyme. The genome of Clostridium thermocellum (ATCC27405 strain), Clostridium cellulolyticum H10 and Clostridium cellulovorans contain 16, 13, and 5 genes belong to GH9 [43]. ...
From an anthropocentric point of view, human culture has been intricately involved in harnessing the potential of lignocellulosic feedstock to bring the bio-competitive alternative of fossil-based fuel resources. In today's scenario, the impact of hyperthermophiles and their enzymes has been intensely investigated for implementation in various high-temperature biotechnological processes. Already characterized archaeal and eubacterial cellulolytic glycoside hydrolase have shown highly impressive catalytic structures and mechanisms. Several sequence and structural factors have simultaneously been proposed to contribute towards the augmented stability of thermophilic proteins. However, state-of-the-art technologies like the rational designing approach and mechanism of directed evolution have emerged as critical toolkits for broadened industrial applications of recombinant proteins. This manuscript discusses the cellulase engineering techniques to enhance the biological production and stability of thermostable cellulolytic enzymes.
... Therefore, the processive glucanases, behaving similarly to exoglucanases, are thought to be a major cellulose-degrading factor in addition to the exoglucanase component [5,6]. A recent study showed that a single GH9 endoglucanase is essential for cellulose degradation by Clostridium phytofermentans, despite its production of several other cellulases [7]. ...
The Clostridium alkalicellulosi DSM17461T genome contains several glucoside hydrolase
encoding genes essential for cellulose degradation. Herein, the family 9 glycoside hydrolase enzyme (CalGH9_2089) was cloned and expressed. The enzyme contains one GH9 catalytic module, a family 3 carbohydrate-binding module, and one Type I dockerin at its C-termini. The optimal pH and temperature for CalGH9_2089 to hydrolyze CMC were 55 ◦C and pH 6.0, with the remaining activity of more than 60% at pH 10.0. CalGH9_2089 produced a series of cello-oligomers (G2-G6) from CMC, suggesting that the enzyme has an endo-acting capability. When regenerated amorphous cellulose was hydrolyzed with CalGH9_2089, the ratio of reducing ends in the soluble fraction to that in the insoluble pellets was 4.8, suggesting that this enzyme acts processively on RAC. This work extends our knowledge of the behavior of the GH9 endoglucanase from the microorganism living in an alkaline environment.
... Real-time PCR amplification was conducted using a Bio-Rad qRT-PCR machine (Hercules, USA) monitoring incorporation of SYBR green I. PCR primers were designed to amplify 100 bp products from the 3 ′ part of each gene. Relative gene expression was quantified using the comparative C T method, and the 16S ribosomal sequence served as an internal control gene (Tolonen et al., 2009). Expression levels were represented as the means of triplicate measurements obtained from each culture. ...
Cellulosomes are scaffold proteins displaying enzymes on the cell wall to efficiently obtain nutrient sources. CcGlcNAcase is a novel cellulosomal component. Based on sequence analysis, CcGlcNAcase was predicted to be a chitinolytic enzyme based on high homology with the discoidin domain-containing protein and chitobiase/ β-hexosaminidase C terminal domain. CcGlcNAcase expression was notably increased when chitin was present. CcGlcNAcase produced N-acetyl-d-glucosamine from various lengths of N-acetyl-d-glucosamine. CcGlcNAcase bound to chitin (89%) and fungi (54.10%), whereas CcGlcNAcase exhibited a low binding ability to cellulose and xylan. CcGlcNAcase hydrolyzed fungi, yielding maximum 3.90 g/L N-acetyl-d-glucosamine. CcGlcNAcase enhanced cellulase toward fungi-infected lignocellulosic biomass, yielding 18 mg/L glucose (1.32-fold) and 1.72-fold increased total reducing sugar levels, whereas cellulase alone produced 13 mg/L glucose. Taken together, CcGlcNAcase can be utilized to enhance the degradation of fungi-infected lignocellulosic biomass and exhibits potential applications in the wood and sugar industry.
... The development of targetrons based on the Lactococcus lactis Ll.LtrB group II intron (9) enabled targeted chromosome insertions in C. phytofermentans (10) and other clostridia (11). Targetrons are designed group II introns that can be customized to insert into specific DNA sequences by a retrohoming mechanism with efficiencies approach-ing 100% of cells (12), obviating the need for antibiotic resistance markers to select for their insertion. ...
... Conjugal transfer of lox71 and lox66 targetron plasmids into C. phytofermentans consistently yielded 10 to 20 transconjugant colonies, of which 80% to 100% contained the expected targetron insertion, indicating that the efficiency of delivery and integration of lox-containing targetrons is similar to that in previous targetrons studies in C. phytofermentans (10,26,27). Following sequential delivery and curing of 2D). ...
... Plasmids were introduced into C. phytofermentans by conjugal transfer from E. coli strain 1100-2 (pRK24) (10), and plasmids were maintained using erythromycin (200 g ml Ϫ1 in liquid medium, 40 g ml Ϫ1 in solid medium) or spectinomycin (600 g ml Ϫ1 in liquid medium and solid medium). Following conjugation, ten transconjugant colonies were picked, and the presence of the plasmid was confirmed by PCR using primers pAMB1_1/2 for pAM1 origin plasmids and primers pBP1_1/2 for pBP1 origin plasmids. ...
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.
... TargeTron technology also are applied in other solventogenic clostridia such as C. phytofermentans [44], C. butylicum [45] and C. tyrobutyricum [46]. Recently, this technology has been extended to cellulolytic clostridia including C. cellulolyticum [47], C. cellulovorans [21,48], and gas-fermenting clostridia C. autoethanogenum [49,50]. ...
Clostridium has great potential in industrial application and medical research. But low DNA repair capacity and plasmids transformation efficiency severely delayed development and application of genetic tools based on homologous recombination (HR). TargeTron is a gene editing technique dependent on the mobility of group II introns, rather than homologous recombination, which made it very suitable for gene disruption of Clostridium. The application of TargeTron technology in Clostridium was academically reported in 2007 and this tool has been introduced in various clostridia as it is easy to operate, time‐saving, and reliable. TargeTron has made great progress in solventogenic Clostridium in the aspects of acetone‐butanol‐ethanol (ABE) fermentation pathway modification, important functional genes identification, and xylose metabolic pathway analysis & reconstruction. In the review, we revisited 12 years’ advances of TargeTron technology applicable in solventogenic Clostridium, including its principle, technical characteristics, application and efforts to expand its capabilities, or to avoid potential drawbacks. Some other technologies as putative competitors or collaborators are also discussed. We believe that TargeTron combined with CRISPR/Cas‐assisted gene/base editing and gene‐expression regulation system will make a better future for clostridial genetic modification. This article is protected by copyright. All rights reserved
... We examine mRNA expression on a panel of plant substrates to identify transporters that are specifically upregulated on hexose substrates. While reverse genetics in Clostridia remain challenging, methods have been developed to make targeted chromosomal insertions using designed group II introns called targetrons (14) in C. phytofermentans (15) and other Clostridia (16). We inactivate the genes encoding these transporters using targetrons to identify which transporters are required for hexose uptake. ...
... The Targetron algorithm (TA0100; Sigma-Aldrich) was used to design primers (see Table S3 in the supplemental material) to insert the intron in the antisense orientation relative to gene transcription at the site closest to the gene start. The targeted intron was PCR amplified and inserted between the NdeI and BsrGI sites of pQint (15). Targeted pQint plasmids were transferred into C. phytofermentans by conjugation with Escherichia coli under anaerobic conditions (15,36,37). ...
... The targeted intron was PCR amplified and inserted between the NdeI and BsrGI sites of pQint (15). Targeted pQint plasmids were transferred into C. phytofermentans by conjugation with Escherichia coli under anaerobic conditions (15,36,37). Colonies on GS2 plates containing the 40 g ml Ϫ1 erythromycin were picked, and the genomic intron insertion was confirmed by PCR and sequencing using primers in Table S3. ...
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.
... In contrast, the cellulolytic and anaerobic bacterium Lachnoclostridium phytofermentans [15] (formerly known as Clostridium phytofermentans [5]) stands as an exception among mesophilic clostridia. Instead of producing extracellular cellulosomes, this microorganism secretes only few cellulases in free state, among which the single GH9 enzyme called Cel9A, drew special attention 10 years ago, as the inactivation of the corresponding gene led to a noncellulolytic strain [16]. The importance of this multimodular enzyme was further confirmed by its characterization [17]: Cel9A exhibits high specific activity on crystalline cellulose far exceeding those reported for all cellulases from R. cellulolyticum characterized to date [14]. ...
Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans are cellulolytic clostridia either producing extracellular multienzymatic complexes termed cellulosomes or secreting free cellulases, respectively. In the free state, the cellulase Cel9A secreted by L. phytofermentans is much more active on crystalline cellulose than any cellulosomal family‐9 enzyme produced by R. cellulolyticum. Nevertheless, the incorporation of Cel9A in vitro in hybrid cellulosomes was formerly shown to generate artificial complexes with altered activity, while its incorporation in vivo in native R. cellulolyticum cellulosomes resulted in a strain displaying a weakened cellulolytic phenotype. In the present study, we investigated why Cel9A is so potent in the free state but functions poorly as a cellulosomal component, in contrast to the most similar enzyme synthesized by R. cellulolyticum, Cel9G, weakly active in the free state but whose activity on crystalline cellulose is drastically increased in cellulosomes. We show that the removal of the C‐terminal moiety of Cel9A encompassing the two X2 modules and the family‐3b cellulose binding module (CBM3b), reduces its activity on crystalline cellulose. Grafting a dockerin module further diminishes the activity, but this truncated cellulosomal form of Cel9A displays important synergies in hybrid cellulosomes with the pivotal family‐48 cellulosomal enzyme of R. cellulolyticum. The exact inverse approach was applied to the cellulosomal Cel9G. Grafting the two X2 modules and the CBM3b of Cel9A to Cel9G strongly increases its activity on crystalline cellulose, to reach Cel9A activity levels. Altogether these data emphasize the specific features required to generate an efficient free or cellulosomal family‐9 cellulase. This article is protected by copyright. All rights reserved.
