scholarly journals Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 996
Author(s):  
Dung Minh Ha-Tran ◽  
Trinh Thi My Nguyen ◽  
Chieh-Chen Huang
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Genetic Material ◽  
Economic Feasibility ◽  
Biofuel Production ◽  
Cellulolytic Bacterium ◽  
Microbial Conversion ◽  
Promising Source

Plant biomass-based biofuels have gradually substituted for conventional energy sources thanks to their obvious advantages, such as renewability, huge quantity, wide availability, economic feasibility, and sustainability. However, to make use of the large amount of carbon sources stored in the plant cell wall, robust cellulolytic microorganisms are highly demanded to efficiently disintegrate the recalcitrant intertwined cellulose fibers to release fermentable sugars for microbial conversion. The Gram-positive, thermophilic, cellulolytic bacterium Clostridium thermocellum possesses a cellulolytic multienzyme complex termed the cellulosome, which has been widely considered to be nature’s finest cellulolytic machinery, fascinating scientists as an auspicious source of saccharolytic enzymes for biomass-based biofuel production. Owing to the supra-modular characteristics of the C. thermocellum cellulosome architecture, the cellulosomal components, including cohesin, dockerin, scaffoldin protein, and the plentiful cellulolytic and hemicellulolytic enzymes have been widely used for constructing artificial cellulosomes for basic studies and industrial applications. In addition, as the well-known microbial workhorses are naïve to biomass deconstruction, several research groups have sought to transform them from non-cellulolytic microbes into consolidated bioprocessing-enabling microbes. This review aims to update and discuss the current progress in these mentioned issues, point out their limitations, and suggest some future directions.

scholarly journals Thermophilic Degradation of Hemicellulose, a Critical Feedstock in the Production of Bioenergy and Other Value-Added Products

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Isaac Cann ◽  
Gabriel V. Pereira ◽  
Ahmed M. Abdel-Hamid ◽  
Heejin Kim ◽  
Daniel Wefers ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Animal Feed ◽  
Value Added ◽  
Building Blocks ◽  
Economic Feasibility ◽  
Renewable Fuels ◽  
Microbial Enzymes

ABSTRACT Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment.

scholarly journals Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation

2021 ◽  
Vol 9 (3) ◽  
pp. 593
Author(s):  
Shunsuke Ichikawa ◽  
Yoichiro Tsuge ◽  
Shuichi Karita
Keyword(s):  
Growth Rate ◽  
Clostridium Thermocellum ◽  
Consolidated Bioprocessing ◽  
Membrane Vesicle ◽  
Single Gene ◽  
Membrane Vesicles ◽  
Growth Stimulation ◽  
Cellulosic Biomass ◽  
Cellulolytic Bacterium ◽  
Metabolome Analysis

The cultivation of the cellulolytic bacterium, Clostridium thermocellum, can have cost-effective cellulosic biomass utilizations, such as consolidated bioprocessing, simultaneous biological enzyme production and saccharification. However, these processes require a longer cultivation term of approximately 1 week. We demonstrate that constituents of the C. thermocellum membrane vesicle fraction significantly promoted the growth rate of C. thermocellum. Similarly, cell-free Bacillus subtilis broth was able to increase C. thermocellum growth rate, while several B. subtilis single-gene deletion mutants, e.g., yxeJ, yxeH, ahpC, yxdK, iolF, decreased the growth stimulation ability. Metabolome analysis revealed signal compounds for cell–cell communication in the C. thermocellum membrane vesicle fraction (ethyl 2-decenoate, ethyl 4-decenoate, and 2-dodecenoic acid) and B. subtilis broth (nicotinamide, indole-3-carboxaldehyde, urocanic acid, nopaline, and 6-paradol). These findings suggest that the constituents in membrane vesicles from C. thermocellum and B. subtilis could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.

scholarly journals Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

2015 ◽  
Vol 81 (16) ◽  
pp. 5567-5573 ◽  
Author(s):  
Scott Wushke ◽  
David B. Levin ◽  
Nazim Cicek ◽  
Richard Sparling
Keyword(s):  
Clostridium Thermocellum ◽  
Type Strain ◽  
Consolidated Bioprocessing ◽  
Lactate Production ◽  
Biofuel Production ◽  
Respiratory Protection ◽  
Facultative Anaerobe ◽  
Experimental Conditions ◽  
Content Type ◽  
Strain Characterization

ABSTRACTDevelopment of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain ofCaldibacillus debilisisolated from an air-tolerant cellulolytic consortium which included aClostridium thermocellumstrain was characterized and compared with theC. debilistype strain. Characterization of isolateC. debilisGB1 and comparisons with the type strain ofC. debilisrevealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobeC. debilisGB1 provided respiratory protection forC. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

scholarly journals The effect of switchgrass plant cell wall properties on its deconstruction by thermochemical pretreatments coupled with fungal enzymatic hydrolysis or Clostridium thermocellum consolidated bioprocessing

2020 ◽  
Vol 22 (22) ◽  
pp. 7924-7945
Author(s):  
Ninad Kothari ◽  
Samarthya Bhagia ◽  
Yunqiao Pu ◽  
Chang Geun Yoo ◽  
Mi Li ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enzymatic Hydrolysis ◽  
Plant Cell ◽  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Consolidated Bioprocessing ◽  
Wall Properties ◽  
Cell Wall Properties

Switchgrass, thermochemically pretreated switchgrass, and corresponding biologically digested residues were characterized to understand the process of lignocelluose deconstruction.

scholarly journals Laboratory evolution and reverse engineering of Clostridium thermocellum for growth on glucose and fructose

2021 ◽  
Author(s):  
Johannes Yayo ◽  
Teun Kuil ◽  
Daniel G. Olson ◽  
Lee R. Lynd ◽  
Evert K. Holwerda ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Molecular Mechanisms ◽  
Consolidated Bioprocessing ◽  
Biofuel Production ◽  
Transport Systems ◽  
Good Growth ◽  
Wild Type ◽  
Laboratory Evolution ◽  
Hexose Sugars

The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 gbiomass gsubstrate−1, respectively, compared to 0.15 ± 0.01 gbiomass gsubstrate−1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, whilst wild type showed 42 ± 4 hours on glucose and >80 hours on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 hours with a growth rate of 0.11 ± 0.01 h−1 and resulted in immediate growth on glucose at 0.24 ± 0.01 h−1. Additional introduction of a cbpAG148V mutation resulted in immediate growth on fructose at 0.32 ± 0.03 h−1. These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings. Importance. C. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to i) reduce glucose accumulation, ii) study cellodextrin transport systems in vivo, iii) allow experiments at >120 g L−1 soluble substrate concentration, or iv) reduce costs for labelling studies.

scholarly journals Compositional and predicted functional analysis of the gut microbiota of Radix auricularia (Linnaeus) via high-throughput Illumina sequencing

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5537 ◽  
Author(s):  
Zongfu Hu ◽  
Xi Chen ◽  
Jie Chang ◽  
Jianhua Yu ◽  
Qing Tong ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Gut Microbiome ◽  
Plant Biomass ◽  
Illumina Miseq ◽  
Biofuel Production ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Bacteria ◽  
Core Microbiome ◽  
Radix Auricularia ◽  
Promising Source

Due to its wide distribution across the world, the snail Radix auricularia plays a central role in the transferal of energy and biomass by consuming plant biomass in freshwater systems. The gut microbiota are involved in the nutrition, digestion, immunity, and development of snails, particularly for cellulolytic bacteria, which greatly contribute to the digestion of plant fiber. For the first time, this study characterized the gut bacterial communities of R. auricularia, as well as predicted functions, using the Illumina Miseq platform to sequence 16S rRNA amplicons. Both juvenile snails (JS) and adult snails (AS) were sampled. The obtained 251,072 sequences were rarefied to 214,584 sequences and clustered into 1,196 operational taxonomic units (OTUs) with 97% sequence identity. The predominant phyla were Proteobacteria (JS: 36.0%, AS: 31.6%) and Cyanobacteria (JS: 16.3%, AS: 19.5%), followed by Chloroflexi (JS: 9.7%, AS: 13.1%), Firmicutes (JS: 14.4%, AS: 6.7%), Actinobacteria (JS: 8.2%, AS: 12.6%), and Tenericutes (JS: 7.3%, AS: 6.2%). The phylum Cyanobacteria may have originated from the plant diet instead of the gut microbiome. A total of 52 bacterial families and 55 genera were found with >1% abundance in at least one sample. A large number of species could not be successfully identified, which could indicate the detection of novel ribotypes or result from insufficient availability of snail microbiome data. The core microbiome consisted of 469 OTUs, representing 88.4% of all sequences. Furthermore, the predicted function of bacterial community of R. auricularia performed by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States suggests that functions related to metabolism and environmental information processing were enriched. The abundance of carbohydrate suggests a strong capability of the gut microbiome to digest lignin. Our results indicate an abundance of bacteria in both JS and AS, and thus the bacteria in R. auricularia gut form a promising source for novel enzymes, such as cellulolytic enzymes, that may be useful for biofuel production. Furthermore, searching for xenobiotic biodegradation bacteria may be a further important application of these snails.

scholarly journals Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Sang-Kyu Jung ◽  
Vinuselvi Parisutham ◽  
Seong Hun Jeong ◽  
Sung Kuk Lee
Keyword(s):  
Cell Wall ◽  
Heterologous Expression ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Host Organism ◽  
Cell Wall Degrading Enzymes ◽  
Degrading Enzymes ◽  
The Cost

A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such asEscherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinantE. coliorZ. mobilisand allow successful consolidated bioprocessing (CBP) in these microorganisms.In-plantaexpression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies.

scholarly journals Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Jingen Li ◽  
Shuying Gu ◽  
Zhen Zhao ◽  
Bingchen Chen ◽  
Qian Liu ◽  
...  
Keyword(s):  
Malic Acid ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Cellulase Activity ◽  
Industrial Applications ◽  
Efficient Production ◽  
Myceliophthora Thermophila ◽  
Final Strain ◽  
Cellobiose Metabolism ◽  
Cost Efficient

Abstract Background Lignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered. Results Using the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207. Conclusions This is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

scholarly journals Feruloyl Esterase Activity of the Clostridium thermocellum Cellulosome Can Be Attributed to Previously Unknown Domains of XynY and XynZ

2000 ◽  
Vol 182 (5) ◽  
pp. 1346-1351 ◽  
Author(s):  
David L. Blum ◽  
Irina A. Kataeva ◽  
Xin-Liang Li ◽  
Lars G. Ljungdahl
Keyword(s):  
Ferulic Acid ◽  
Esterase Activity ◽  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Feruloyl Esterase ◽  
Lower Amount ◽  
Acetyl Xylan Esterase ◽  
Hydrolytic Activities ◽  
C And N ◽  
Cellulose Binding

ABSTRACT The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, β-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungusOrpinomyces. Purified cellulosomes from C. thermocellum were found to hydrolyze FAXX (O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose) and FAX3(5-O-[(E)-feruloyl]-[O-β-d-xylopyranosyl-(1→2)]-O-α-l-arabinofuranosyl-[1→3]}-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose), yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia coli, and the expressed proteins hydrolyzed FAXX and FAX3. The recombinant feruloyl esterase domain of XynZ alone (FAEXynZ) and with the adjacent cellulose binding domain (FAE-CBDXynZ) were characterized. FAE-CBDXynZhad a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. Km andV max values for FAX3 were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60°C. PAX3, a substrate similar to FAX3 but with ap-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70°C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount ofp-coumaric acid. FAEXynZ had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAEXynZ resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the C. thermocellumcellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.

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