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Biotechnology For Biofuels 2015Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are...
BACKGROUND
Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are anaerobic bacteria that developed different strategies to depolymerize the cellulose and the related plant cell wall polysaccharides. Thus, R. cellulolyticum produces large extracellular multi-enzyme complexes termed cellulosomes, while L. phytofermentans secretes in the environment some cellulose-degrading enzymes as free enzymes. In the present study, the major cellulase from L. phytofermentans was introduced as a free enzyme or as a cellulosomal component in R. cellulolyticum to improve its cellulolytic capacities.
RESULTS
The gene at locus Cphy_3367 encoding the major cellulase Cel9A from L. phytofermentans and an engineered gene coding for a modified enzyme harboring a R. cellulolyticum C-terminal dockerin were cloned in an expression vector. After electrotransformation of R. cellulolyticum, both forms of Cel9A were found to be secreted by the corresponding recombinant strains. On minimal medium containing microcrystalline cellulose as the sole source of carbon, the strain secreting the free Cel9A started to grow sooner and consumed cellulose faster than the strain producing the cellulosomal form of Cel9A, or the control strain carrying an empty expression vector. All strains reached the same final cell density but the strain producing the cellulosomal form of Cel9A was unable to completely consume the available cellulose even after an extended cultivation time, conversely to the two other strains. Analyses of their cellulosomes showed that the engineered form of Cel9A bearing a dockerin was successfully incorporated in the complexes, but its integration induced an important release of regular cellulosomal components such as the major cellulase Cel48F, which severely impaired the activity of the complexes on cellulose. In contrast, the cellulosomes synthesized by the control and the free Cel9A-secreting strains displayed similar composition and activity. Finally, the most cellulolytic strain secreting free Cel9A, was also characterized by an early production of lactate, acetate and ethanol as compared to the control strain.
CONCLUSIONS
Our study shows that the cellulolytic capacity of R. cellulolyticum can be augmented by supplementing the cellulosomes with a free cellulase originating from L. phytofermentans, whereas integration of the heterologous enzyme in the cellulosomes is rather unfavorable.
PubMed: 26269713
DOI: 10.1186/s13068-015-0301-4 -
FEMS Microbiology Reviews May 2014Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes... (Review)
Review
Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.
Topics: Biofuels; Cellulosomes; Gram-Positive Endospore-Forming Bacteria; Hot Temperature; Lignin; Molecular Sequence Data; Plants; Soil Microbiology
PubMed: 24118059
DOI: 10.1111/1574-6976.12044 -
Microbial Biotechnology Jul 2021Health depends on the diet and a vegetal diet promotes health by providing fibres, vitamins and diverse metabolites. Remarkably, plants may also provide microbes. Fungi... (Review)
Review
Health depends on the diet and a vegetal diet promotes health by providing fibres, vitamins and diverse metabolites. Remarkably, plants may also provide microbes. Fungi and bacteria that reside inside plant tissues (endophytes) seem better protected to survive digestion; thus, we investigated the reported evidence on the endophytic origin of some members of the gut microbiota in animals such as panda, koala, rabbits and tortoises and several herbivore insects. Data examined here showed that some members of the herbivore gut microbiota are common plant microbes, which derived to become stable microbiota in some cases. Endophytes may contribute to plant fibre or antimetabolite degradation and synthesis of metabolites with the plethora of enzymatic activities that they display; some may have practical applications, for example, Lactobacillus plantarum found in the intestinal tract, plants and in fermented food is used as a probiotic that may defend animals against bacterial and viral infections as other endophytic-enteric bacteria do. Clostridium that is an endophyte and a gut bacterium has remarkable capabilities to degrade cellulose by having cellulosomes that may be considered the most efficient nanomachines. Cellulose degradation is a challenge in animal digestion and for biofuel production. Other endophytic-enteric bacteria may have cellulases, pectinases, xylanases, tannases, proteases, nitrogenases and other enzymatic capabilities that may be attractive for biotechnological developments, indeed many endophytes are used to promote plant growth. Here, a cycle of endophytic-enteric-soil-endophytic microbes is proposed which has relevance for health and comprises the fate of animal faeces as natural microbial inoculants for plants that constitute bacterial sources for animal guts.
Topics: Animals; Endophytes; Fungi; Herbivory; Plant Development; Plants
PubMed: 33320440
DOI: 10.1111/1751-7915.13688 -
Applied Microbiology and Biotechnology Sep 2019The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the... (Comparative Study)
Comparative Study Review
The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism's fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.
Topics: Animals; Biological Evolution; Biopolymers; Cellulosomes; Fungal Proteins; Fungi; Genome, Fungal; Phylogeny; Recombinant Proteins; Rumen
PubMed: 31309267
DOI: 10.1007/s00253-019-09983-w -
Bioengineered May 2017The evolution of natural modular proteins and domain swapping by protein engineers have shown the disruptive potential of non-homologous recombination to create proteins... (Review)
Review
The evolution of natural modular proteins and domain swapping by protein engineers have shown the disruptive potential of non-homologous recombination to create proteins with novel functions or traits. Bacteriophage endolysins, cellulosomes and polyketide synthases are 3 examples of natural modular proteins with each module having a dedicated function. These modular architectures have been created by extensive duplication, shuffling of domains and insertion/deletion of new domains. Protein engineers mimic these natural processes in vitro to create chimeras with altered properties or novel functions by swapping modules between different parental genes. Most domain swapping efforts are realized with traditional restriction and ligation techniques, which become particularly restrictive when either a large number of variants, or variants of proteins with multiple domains have to be constructed. Recent advances in homology-independent shuffling techniques increasingly address this need, but to realize the full potential of the synthetic biology of modular proteins a complete homology-independent method for both rational and random shuffling of modules from an unlimited number of parental genes is still needed.
Topics: Biomimetic Materials; Biosynthetic Pathways; Enzyme Activation; Enzymes; Protein Engineering; Substrate Specificity; Synthetic Biology
PubMed: 27645260
DOI: 10.1080/21655979.2016.1222993 -
International Journal of Biological... Sep 2009The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and... (Review)
Review
The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and beta-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.
Topics: Biomass; Cellulase; Cellulosomes; Fungal Proteins; Fungi; Lignin
PubMed: 19774110
DOI: 10.7150/ijbs.5.578 -
MBio Oct 2022Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an...
Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates. Here, we first applied combined genetic manipulations and biophysical titration experiments to decipher the key glucose and cellodextrin transporters. gene inactivation of each transporter and calorimetric and nuclear magnetic resonance (NMR) titration of each putative sugar-binding protein with various saccharides supported the conclusion that only transporters A and B play the roles of glucose and cellodextrin transport, respectively. To gain insight into the structural mechanism of the transporter specificities, 11 crystal structures, both alone and in complex with appropriate saccharides, were solved for all 5 putative sugar-binding proteins, thus providing detailed specific interactions between the proteins and the corresponding saccharides. Considering the importance of transporter B as the major cellodextrin transporter, we further identified its cryptic, hitherto unknown ATPase-encoding gene as , which is located outside the transporter B gene cluster. The crystal structure of the ATPase was solved, showing that it represents a typical nucleotide-binding domain of the ATP-binding cassette (ABC) transporter. Moreover, we determined that the inducing effect of cellobiose (G2) and cellulose on cellulosome production could be eliminated by deletion of transporter B genes, suggesting the coupling of sugar transport and regulation of cellulosome components. This study provides key basic information on the sugar uptake mechanism of and will promote rational engineering of the bacterium for industrial application. Highly efficient sugar uptake is important to microbial cell factories, and sugar transporters are therefore of great interest in the study of industrially relevant microorganisms. Clostridium thermocellum is a lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery. In this study, we clarify the function and mechanism of substrate specificity of the five reported putative sugar transporters using genetic, biophysical, and structural methods. Intriguingly, the results showed that only one of them, transporter B, is the major cellodextrin transporter, whereas another, transporter A, represents the major glucose transporter. Considering the importance of transporter B, we further identified the missing ATPase gene of transporter B and revealed the correlation between transporter B and cellulosome production. Revealing the mechanism by which utilizes cellodextrins will help pave the way for engineering the strain for industrial applications.
Topics: Clostridium thermocellum; Cellobiose; Cellulose; ATP-Binding Cassette Transporters; Glucose; Adenosine Triphosphatases; Glucose Transport Proteins, Facilitative; Nucleotides; Adenosine Triphosphate
PubMed: 36069444
DOI: 10.1128/mbio.01476-22 -
Bioengineered 2015Extraction of sugar is the rate-limiting step in converting unpretreated biomass into value-added products through microbial fermentation. Both anaerobic fungi and... (Review)
Review
Extraction of sugar is the rate-limiting step in converting unpretreated biomass into value-added products through microbial fermentation. Both anaerobic fungi and anaerobic bacteria have evolved to produce large multi-cellulase complexes referred to as cellulosomes, which are powerful machines for biomass deconstruction. Characterization of bacterial cellulosomes has inspired synthetic "designer" cellulosomes, consisting of parts discovered from the native system that have proven useful for cellulose depolymerization. By contrast, the multi-cellulase complexes produced by anaerobic fungi are much more poorly understood, and to date their composition, architecture, and enzyme tethering mechanism remain unknown and heavily debated. Here, we compare current knowledge pertaining to the cellulosomes produced by both bacteria and fungi, including their application to synthetic enzyme-tethered systems for tunneled biocatalysis. We highlight gaps in knowledge and opportunities for discovery, especially pertaining to the potential of fungal cellulosome-inspired systems.
Topics: Bacteria; Bacterial Proteins; Biocatalysis; Bioengineering; Biomass; Cellulases; Cellulose; Cellulosomes; Fermentation; Fungal Proteins; Fungi; Glucose; Glycoside Hydrolases; Hydrolysis; Multienzyme Complexes
PubMed: 26068180
DOI: 10.1080/21655979.2015.1060379 -
Environmental Research Jul 2023Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment... (Review)
Review
Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment for the storage and microbial fermentation of ingested feeds dominated by plant materials. The rumen microbiota has acquired diverse and functionally overlapped enzymes for the degradation of plant cell wall polysaccharides. In rumen Bacteroidetes, enzymes involved in degradation are clustered into polysaccharide utilization loci to facilitate coordinated expression when target polysaccharides are available. Firmicutes use free enzymes and cellulosomes to degrade the polysaccharides. Fibrobacters either aggregate lignocellulose-degrading enzymes on their cell surface or release them into the extracellular medium in membrane vesicles, a mechanism that has proven extremely effective in the breakdown of recalcitrant cellulose. Based on current metagenomic analyses, rumen Bacteroidetes and Firmicutes are categorized as generalist microbes that can degrade a wide range of polysaccharides, while other members adapted toward specific polysaccharides. Particularly, there is ample evidence that Verrucomicrobia and Spirochaetes have evolved enzyme systems for the breakdown of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin. It is concluded that diversity in degradation mechanisms is required to ensure that every component in feeds is efficiently degraded, which is key to harvesting maximum energy by host animals.
Topics: Animals; Metagenome; Rumen; Lignin; Bacteria; Polysaccharides; Bacteroidetes
PubMed: 37086884
DOI: 10.1016/j.envres.2023.115925 -
MBio Jun 2021Anaerobic fungi () isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of...
Anaerobic fungi () isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. Anaerobic fungi () isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.
Topics: Anaerobiosis; Biomass; Cellulosomes; Hydrolysis; Piromyces
PubMed: 34061594
DOI: 10.1128/mBio.00832-21