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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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... The expected intron insertion, 37 bp downstream of the start codon cphy3367 in the antisense direction (Fig. 1B), was confirmed by PCR mapping of the cphy3367 locus in the AT02 isolates. PCR of the cphy3367 coding region yielded a 3.1 kb product in wild type and a 4 kb product in AT02-1, supporting the expected 0.9 kb intron insertion in cphy3367 (Fig. 3A, lanes 1 and 2). PCR of the 5′ and the 3′ intron-genome junction regions using one primer in the genome and the other in the intron (3367_F/Ebs2_3367 for the 5′ junction and 3367_R/Ebs_univ for the 3′ junction) resulted in prod- ucts in AT02-1 ( Fig. 3A, lanes 4 and 6), but not wild type ( Fig. 3A, lanes 3 and 5). These PCR products ...
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... and a 4 kb product in AT02-1, supporting the expected 0.9 kb intron insertion in cphy3367 (Fig. 3A, lanes 1 and 2). PCR of the 5′ and the 3′ intron-genome junction regions using one primer in the genome and the other in the intron (3367_F/Ebs2_3367 for the 5′ junction and 3367_R/Ebs_univ for the 3′ junction) resulted in prod- ucts in AT02-1 ( Fig. 3A, lanes 4 and 6), but not wild type ( Fig. 3A, lanes 3 and 5). These PCR products were sequenced to confirm that AT02-1 bears the expected intron insertion in cphy3367 (Fig. S1). Similarly, PCR screening of the other nine AT02 isolates confirmed the correct insertion in cphy3367, supporting that the A. PCR of the cphy3367 locus in ...
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... the expected 0.9 kb intron insertion in cphy3367 (Fig. 3A, lanes 1 and 2). PCR of the 5′ and the 3′ intron-genome junction regions using one primer in the genome and the other in the intron (3367_F/Ebs2_3367 for the 5′ junction and 3367_R/Ebs_univ for the 3′ junction) resulted in prod- ucts in AT02-1 ( Fig. 3A, lanes 4 and 6), but not wild type ( Fig. 3A, lanes 3 and 5). These PCR products were sequenced to confirm that AT02-1 bears the expected intron insertion in cphy3367 (Fig. S1). Similarly, PCR screening of the other nine AT02 isolates confirmed the correct insertion in cphy3367, supporting that the A. PCR of the cphy3367 locus in wild-type and AT02-1 strains. The cphy3367 gene in ...
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... 1.0) through five serial transfers. AT02-1 was then plated on solid GS2 (-erm) medium and 10 colonies were tested by PCR, both for the presence of the erm R gene in pQint3367 and for the retention of an intron insertion in cphy3367 with primers 3367_F and Ebs2_3367. Transfer in medium lacking erythromycin resulted in rapid loss of pQint3367 (Fig. 3B). In total, 8 of 10 colonies had lost pQint3367, while all 10 had retained the intron insertion in cphy3367 (Fig. S2A). Further, plating on GS2 medium containing erythromycin showed that strains that had lost pQint3367 were erythromycin-sensitive (Fig. S2B). After an addi- tional five transfers in the absence of erythromycin selec- ...
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... blotting showed that AT02-1 and AT02-2 do not harbour any additional intron insertions elsewhere in the genome (Fig. 3C). The Southern blot showed no bands in wild type (Fig. 3C, lane 1) and a single band in both transconjugants, AT02-1 and AT02-2, at the expected size of 5855 bp (Fig. 3C, lanes 2 and 3). A Southern blot of AT02-1 prior to loss of pQint3367 showed a second band of 7799 bp due to the plasmid-born intron. The Ll.LtrB group II intron can ...
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... blotting showed that AT02-1 and AT02-2 do not harbour any additional intron insertions elsewhere in the genome (Fig. 3C). The Southern blot showed no bands in wild type (Fig. 3C, lane 1) and a single band in both transconjugants, AT02-1 and AT02-2, at the expected size of 5855 bp (Fig. 3C, lanes 2 and 3). A Southern blot of AT02-1 prior to loss of pQint3367 showed a second band of 7799 bp due to the plasmid-born intron. The Ll.LtrB group II intron can thus be used for targeted gene disruptions in C. phytofermentans ...
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... blotting showed that AT02-1 and AT02-2 do not harbour any additional intron insertions elsewhere in the genome (Fig. 3C). The Southern blot showed no bands in wild type (Fig. 3C, lane 1) and a single band in both transconjugants, AT02-1 and AT02-2, at the expected size of 5855 bp (Fig. 3C, lanes 2 and 3). A Southern blot of AT02-1 prior to loss of pQint3367 showed a second band of 7799 bp due to the plasmid-born intron. The Ll.LtrB group II intron can thus be used for targeted gene disruptions in C. phytofermentans without making additional, unintended genomic ...
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... that the erm R gene from S. pneumoniae Tn1545 gives high erythromycin resistance in this organism. Further, the Enterococcus pAMb1 origin of replication replicates stably in C. phytofermentans under antibiotic selection, but can be easily cured from strains in the absence of selection. Finally, the L. lactis Ll.LtrB group II intron makes accurate (Fig. 3A) and specific (Fig. 3C) chromosomal insertions in C. phytofermentans. We found that when the strong C. phytofermentans cphy3558 promoter is used to drive expression of the intron, it inserts into the genome with such a high efficiency that mutants can be easily isolated without selecting for integration. As the intron insertions are ...
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... S. pneumoniae Tn1545 gives high erythromycin resistance in this organism. Further, the Enterococcus pAMb1 origin of replication replicates stably in C. phytofermentans under antibiotic selection, but can be easily cured from strains in the absence of selection. Finally, the L. lactis Ll.LtrB group II intron makes accurate (Fig. 3A) and specific (Fig. 3C) chromosomal insertions in C. phytofermentans. We found that when the strong C. phytofermentans cphy3558 promoter is used to drive expression of the intron, it inserts into the genome with such a high efficiency that mutants can be easily isolated without selecting for integration. As the intron insertions are stable in the absence of ...
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... The GH9-deficient strain, AT02-1, grows normally on glucose, cellobiose and hemicellulose (Fig. 4A-C) but has lost the ability to degrade crystalline cellulose ( Fig. 4D and E). Several lines of evidence support that this phenotype resulted specifically from the inactivation of Cphy3367. AT02-1 did not have any additional intron insertions (Fig. 3C). The gene expression patterns of cphy3367 and surrounding genes (Fig. 5) support that cphy3367 is the downstream member of a two-gene operon such that inactivation of cphy3367 would not result in polar transcriptional effects. Finally, the expression of CAZy genes in wild type and AT02-1 appears similar, supporting that disruption of ...
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Citations
... Early studies found that conjugative transposons bearing antibiotic resistance genes transfer DNA between Lachnospiraceae ( Barbosa et al., 1999). Experimental methods have been developed to transfer plasmid DNA into species of Lachnoclostridium, Roseburia, Eubacterium, Enterocloster, Lacrimispora, and Blautia by conjugation with Escherichia coli (Tolonen et al., 2009;Cuív et al., 2015;Sheridan et al., 2019;Jin et al., 2022) and by electroporation into species of Lachnoclostridium and Butyrivibrio (Beard et al., 1995;Rostain et al., 2022) (Figure 4A). ...
... Similar to other Clostridia, low rates of DNA transfer and homologous recombination in Lachnospiraceae have led to the use of other recombination systems to make targeted chromosomal changes. Designed group II intron called targetrons enabled gene inactivation by targeted chromosome insertion in various Lachnospiraceae with efficiencies ranging from 12.5%-100% (Tolonen et al., 2009;Tolonen et al., 2015a;Cerisy et al., 2019a;Jin et al., 2022) (Figure 4D). Multi-gene fragments can be excised and inserted by modifying targetrons to deliver lox sites into the genome that act as anchor points for Cre-mediated recombination, which has been applied to delete a 39 kb prophage in L. phytofermentans (Cerisy et al., 2019b). ...
The Lachnospiraceae is a family of anaerobic bacteria in the class Clostridia with potential to advance the bio-economy and intestinal therapeutics. Some species of Lachnospiraceae metabolize abundant, low-cost feedstocks such as lignocellulose and carbon dioxide into value-added chemicals. Others are among the dominant species of the human colon and animal rumen, where they ferment dietary fiber to promote healthy gut and immune function. Here, we summarize recent studies of the physiology, cultivation, and genetics of Lachnospiraceae, highlighting their wide substrate utilization and metabolic products with industrial applications. We examine studies of these bacteria as Live Biotherapeutic Products (LBPs), focusing on in vivo disease models and clinical studies using them to treat infection, inflammation, metabolic syndrome, and cancer. We discuss key research areas including elucidation of intra-specific diversity and genetic modification of candidate strains that will facilitate the exploitation of Lachnospiraceae in industry and medicine.
... 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.
... 27−31 Conjugation showed that pQexp, which bears the pAMβ1 replicon and erythromycin resistance gene, stably replicates C. phytofermentans. 27 To develop methods for electrotransformation of C. phytofermentans, we evaluated the effects of electropulse, DNA concentration, and cell wall-weakening osmolytes on electroporation of pQexp using an exponential-decay wave pulse. All electroporations were performed at the bench without an anaerobic glovebox, making these methods generally accessible to microbiology laboratories. ...
... The vector backbone to express the NanoLuc gene was built by PCR amplification of the erm gene (primers PR3/PR4), pAMβ1 replicon (primers PR5/PR6), and pUC replicon (primers PR7/PR8) of pQexp. 27 The fragments were assembled with the NanoLuc PCR fragment by four-part Golden Gate cloning using BsmBI, yielding pQnl_Pcons17 (Table 1). pATmin-GG was constructed by Gibson assembly from PCR of pQnl_Pcons17 with PR70/PR71 and of pQmod2C-GG with PR72/PR73. ...
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.
... 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
