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 then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367.
... 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.
... 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.
... 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.
... 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.
... 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.
... 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 base organism for our effort is Clostridium phytofermentans (Cphy), a gene5cally tractable model bacterium that can directly convert a broad range of biomass sources into ethanol and hydrogen gas 1 . One of the most aerac5ve characteris5cs of this organism is that Cphy can produce biofuels without the need for expensive thermochemical pretreatment and enzyme addi5ons [2][3][4] . Here we report the latest results of our engineering effort. ...
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.
Background
Environmental bacteria express a wide diversity of glycoside hydrolases (GH). Screening and characterization of GH from metagenomic sources provides an insight into biomass degradation strategies of non-cultivated prokaryotes.
Methods
In the present report, we screened a compost metagenome for lignocellulolytic activities and identified six genes encoding enzymes belonging to family GH9 (GH9a-f). Three of these enzymes (GH9b, GH9d and GH9e) were successfully expressed and characterized.
Results
A phylogenetic analysis of the catalytic domain of pro- and eukaryotic GH9 enzymes suggested the existence of two major subgroups. Bacterial GH9s displayed a wide variety of modular architectures and those harboring an N-terminal Ig-like domain, such as GH9b and GH9d, segregated from the remainder. We purified and characterized GH9 endoglucanases from both subgroups and examined their stabilities, substrate specificities and product profiles. GH9e exhibited an original hydrolysis pattern, liberating an elevated proportion of oligosaccharides longer than cellobiose. All of the enzymes exhibited processive behavior and a synergistic action on crystalline cellulose. Synergy was also evidenced between GH9d and a GH48 enzyme identified from the same metagenome.
Conclusions
The characterized GH9 enzymes displayed different modular architectures and distinct substrate and product profiles. The presence of a cellulose binding domain was shown to be necessary for binding and digestion of insoluble cellulosic substrates, but not for processivity.
General significance
The identification of six GH9 enzymes from a compost metagenome and the functional variety of three characterized members highlight the importance of this enzyme family in bacterial biomass deconstruction.
The U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA) are both strongly committed to expanding the role of biomass as an energy source. In particular, they support biomass fuels and products as a way to reduce the need for oil and gas imports; to support the growth of agriculture, forestry, and rural economies; and to foster major new domestic industries-- biorefineries--making a variety of fuels, chemicals, and other products. As part of this effort, the Biomass R&D Technical Advisory Committee, a panel established by the Congress to guide the future direction of federally funded biomass R&D, envisioned a 30 percent replacement of the current U.S. petroleum consumption with biofuels by 2030. Biomass--all plant and plant-derived materials including animal manure, not just starch, sugar, oil crops already used for food and energy--has great potential to provide renewable energy for America s future. Biomass recently surpassed hydropower as the largest domestic source of renewable energy and currently provides over 3 percent of the total energy consumption in the United States. In addition to the many benefits common to renewable energy, biomass is particularly attractive because it is the only current renewable source of liquid transportation fuel. This, of course, makes it invaluable in reducing oil imports--one of our most pressing energy needs. A key question, however, is how large a role could biomass play in responding to the nation's energy demands. Assuming that economic and financial policies and advances in conversion technologies make biomass fuels and products more economically viable, could the biorefinery industry be large enough to have a significant impact on energy supply and oil imports? Any and all contributions are certainly needed, but would the biomass potential be sufficiently large to justify the necessary capital replacements in the fuels and automobile sectors?
The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown
complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside
hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families.
These families are created based on experimentally characterized proteins and are populated by sequences from public databases
with significant similarity. Protein biochemical information is continuously curated based on the available literature and
structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification
(i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the
evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties.
This resource has been available for over 10 years to the scientific community, contributing to information dissemination
and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality
of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/.
Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative CT method also referred to as the 2−ΔΔCT method. This protocol provides an overview of the comparative CT method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.
Endoglucanase A (CenA) from the bacterium Cellulomonas fimi is composed of a catalytic domain and a nonhydrolytic cellulose-binding domain that can function independently. The individual domains interact synergistically in the disruption and hydrolysis of cellulose fibers. This intramolecular synergism is distinct from the well-known intermolecular synergism between individual cellulases. The catalytic domain corresponds to the hydrolytic C_x system and the cellulose-binding domain corresponds to the nonhydrolytic C_1 system postulated by Reese et al. [Reese, E. T., Sui, R. G. H. & Levinson, H. S. (1950) J. Bacteriol. 59, 485-497] to be required for the hydrolysis of cellulose.
The nucleotide sequence of the celZ gene coding for a thermostable endo--1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 by which encodes a preprotein of 986 amino acids with an Mr of 109000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fuorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.
A method to correlate the uninterpreted tandem mass spectra of peptides produced under low energy (10–50 eV) collision conditions with amino acid sequences in the Genpept database has been developed. In this method the protein database is searched to identify linear amino acid sequences within a mass tolerance of ± 1 u of the precursor ion molecular weight. A cross-correlation function is then used to provide a measurement of similarity between the mass-to-charge ratios for the fragment ions predicted from amino acid sequences obtained from the database and the fragment ions observed in the tandem mass spectrum. In general, a difference greater than 0.1 between the normalized cross-correlation functions of the first- and second-ranked search results indicates a successful match between sequence and spectrum. Searches of species-specific protein databases with tandem mass spectra acquired from peptides obtained from the enzymatically digested total proteins of E. coli and S. cerevisiae cells allowed matching of the spectra to amino acid sequences within proteins of these organisms. The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.
A chromosomally integrated copy of the IncP plasmid RP4 was shown to mediate the conjugative transfer of shuttle plasmids containing an oriT site from Escherichia coli to the anaerobic pathogen Clostridium perfringens. Two versatile shuttle plasmids, pJIR1456 and pJIR1457, which will be invaluable for the introduction of cloned genes into C. perfringens, were constructed.
The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, BamH-I, SalI and HpaI. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.
