scholarly journals Expression of a Cellobiose Phosphorylase fromThermotoga maritimainCaldicellulosiruptor besciiImproves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Sun-Ki Kim ◽  
Michael E. Himmel ◽  
Yannick J. Bomble ◽  
Janet Westpheling
Keyword(s):  
Extracellular Enzymes ◽  
Plant Biomass ◽  
Rational Approach ◽  
Crystalline Cellulose ◽  
Synergistic Activity ◽  
Cellulolytic Activity ◽  
Glycosyl Hydrolases ◽  
Content Type ◽  
Biomass Deconstruction ◽  
Cellobiose Phosphorylase

ABSTRACTMembers of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase fromThermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, including CelA, to increase cellulolytic activity and growth on crystalline cellulose.IMPORTANCECelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. This work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.

scholarly journals ABC Transporters Required for Hexose Uptake byClostridium phytofermentans

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Tristan Cerisy ◽  
Alba Iglesias ◽  
William Rostain ◽  
Magali Boutard ◽  
Christine Pelle ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Extracellular Enzymes ◽  
Plant Biomass ◽  
Carbon Sources ◽  
Anaerobic Bacteria ◽  
Model Organism ◽  
Industrial Transformation ◽  
Content Type ◽  
Plant Matter

ABSTRACTThe mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal howClostridium(Lachnoclostridium)phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based onin vitroactivities. These mechanisms used byClostridiato uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCEPlant-fermentingClostridiaare anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine.Clostridiadegrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organismClostridium phytofermentans. We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

scholarly journals Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass

2014 ◽  
Vol 80 (23) ◽  
pp. 7423-7432 ◽  
Author(s):  
Stephanie A. Eichorst ◽  
Chijioke Joshua ◽  
Noppadon Sathitsuksanoh ◽  
Seema Singh ◽  
Blake A. Simmons ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Plant Biomass ◽  
Biofuel Production ◽  
Rrna Gene ◽  
Gravimetric Analysis ◽  
Content Type ◽  
Biomass Deconstruction ◽  
Bacterial Consortia ◽  
Residual Biomass ◽  
Chemical Pretreatments

ABSTRACTMicrobial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of theFirmicutes, theBacteroidetes, andDeinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction.

Predominant secretion of cellobiohydrolases and endo-β-1,4-glucanases in nutrient-limited medium by Aspergillus spp. isolated from subtropical field

2020 ◽  
Vol 168 (3) ◽  
pp. 243-256 ◽  
Author(s):  
May Thin Kyu ◽  
Shunsuke Nishio ◽  
Koki Noda ◽  
Bay Dar ◽  
San San Aye ◽  
...  
Keyword(s):  
Rice Straw ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Industrial Wastes ◽  
Added Value ◽  
Crystalline Cellulose ◽  
Cellulolytic Activity ◽  
Biological Degradation ◽  
Industry Waste ◽  
Degradation Intermediates

Abstract Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50–60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-β-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-β-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

scholarly journals S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

2011 ◽  
Vol 78 (3) ◽  
pp. 768-777 ◽  
Author(s):  
Inci Ozdemir ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly
Keyword(s):  
Cellular Localization ◽  
Catalytic Domain ◽  
Plant Biomass ◽  
Thermophilic Bacteria ◽  
Biochemical Properties ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Content Type

ABSTRACTThe genusCaldicellulosiruptorcontains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes ofCaldicellulosiruptorspecies reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins inCaldicellulosiruptor saccharolyticus(Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequencedCaldicellulosiruptorspecies. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in theC. saccharolyticusgenome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to theC. saccharolyticusS-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in otherCaldicellulosiruptorgenomes may also be important contributors to plant biomass utilization.

scholarly journals Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Narumon Tangthirasunun ◽  
David Navarro ◽  
Sona Garajova ◽  
Didier Chevret ◽  
Laetitia Chan Ho Tong ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Podospora Anserina ◽  
Cellulosic Biomass ◽  
Striking Difference ◽  
Crystalline Cellulose ◽  
Minor Role ◽  
Wild Type ◽  
Content Type

ABSTRACT Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

scholarly journals Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Jordan F. Russell ◽  
Matthew L. Russo ◽  
Xuewen Wang ◽  
Neal Hengge ◽  
Daehwan Chung ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Plant Biomass ◽  
Protein Glycosylation ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Unique Gene ◽  
Glycosyl Transferase ◽  
Caldicellulosiruptor Bescii ◽  
Carbohydrate Binding Modules

ABSTRACT Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases. IMPORTANCE Caldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

scholarly journals Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions

2013 ◽  
Vol 79 (17) ◽  
pp. 5242-5249 ◽  
Author(s):  
Sarah Moraïs ◽  
Naama Shterzer ◽  
Inna Rozman Grinberg ◽  
Geir Mathiesen ◽  
Vincent G. H. Eijsink ◽  
...  
Keyword(s):  
Wheat Straw ◽  
Lactobacillus Plantarum ◽  
Plant Biomass ◽  
Soluble Sugar ◽  
Acid Tolerance ◽  
Cellulolytic Bacterium ◽  
Synergistic Activity ◽  
Heterologous Proteins ◽  
Content Type ◽  
Highly Active

ABSTRACTLactobacillus plantarumis an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium intoL. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted byL. plantarumat levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD600) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing ofL. plantarumfor novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.

scholarly journals Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose

2011 ◽  
Vol 77 (8) ◽  
pp. 2727-2733 ◽  
Author(s):  
Wendy Higashide ◽  
Yongchao Li ◽  
Yunfeng Yang ◽  
James C. Liao
Keyword(s):  
Metabolic Engineering ◽  
Consolidated Bioprocessing ◽  
Cellulose Degradation ◽  
Crystalline Cellulose ◽  
Cellulolytic Activity ◽  
Keto Acid ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Clostridium Cellulolyticum ◽  
Alcohol Synthesis

ABSTRACTProducing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example for the development of aClostridium cellulolyticumstrain for isobutanol synthesis directly from cellulose. This strategy exploits the host's natural cellulolytic activity and the amino acid biosynthesis pathway and diverts its 2-keto acid intermediates toward alcohol synthesis. Specifically, we have demonstrated the first production of isobutanol to approximately 660 mg/liter from crystalline cellulose by using this microorganism.

scholarly journals Caldicellulosiruptor bescii Adheres to Polysaccharides via a Type IV Pilin-Dependent Mechanism

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Asma M. A. M. Khan ◽  
Valerie J. Hauk ◽  
Mena Ibrahim ◽  
Thomas R. Raffel ◽  
Sara E. Blumer-Schuette
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Elevated Temperatures ◽  
Plant Biomass ◽  
Cell Attachment ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Iv

ABSTRACT Biological hydrolysis of cellulose above 70°C involves microorganisms that secrete free enzymes and deploy separate protein systems to adhere to their substrate. Strongly cellulolytic Caldicellulosiruptor bescii is one such extreme thermophile, which deploys modular, multifunctional carbohydrate-acting enzymes to deconstruct plant biomass. Additionally, C. bescii also encodes noncatalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in carbon-limited biotopes that these bacteria inhabit. Analysis of the Caldicellulosiruptor pangenome identified a type IV pilus (T4P) locus encoded upstream of the tāpirins, that is encoded by all Caldicellulosiruptor species. In this study, we sought to determine if the C. bescii T4P plays a role in attachment to plant polysaccharides. The major C. bescii pilin (CbPilA) was identified by the presence of pilin-like protein domains, paired with transcriptomics and proteomics data. Using immuno-dot blots, we determined that the plant polysaccharide xylan induced production of CbPilA 10- to 14-fold higher than glucomannan or xylose. Furthermore, we are able to demonstrate that recombinant CbPilA directly interacts with xylan and cellulose at elevated temperatures. Localization of CbPilA at the cell surface was confirmed by immunofluorescence microscopy. Lastly, a direct role for CbPilA in cell adhesion was demonstrated using recombinant CbPilA or anti-CbPilA antibodies to reduce C. bescii cell adhesion to xylan and crystalline cellulose up to 4.5- and 2-fold, respectively. Based on these observations, we propose that CbPilA and, by extension, the T4P play a role in Caldicellulosiruptor cell attachment to plant biomass. IMPORTANCE Most microorganisms are capable of attaching to surfaces in order to persist in their environment. Type IV (T4) pili produced by certain mesophilic Firmicutes promote adherence; however, a role for T4 pili encoded by thermophilic members of this phylum has yet to be demonstrated. Prior comparative genomics analyses identified a T4 pilus locus possessed by an extremely thermophilic genus within the Firmicutes. Here, we demonstrate that attachment to plant biomass-related carbohydrates by strongly cellulolytic Caldicellulosiruptor bescii is mediated by T4 pilins. Surprisingly, xylan but not cellulose induced expression of the major T4 pilin. Regardless, the C. bescii T4 pilin interacts with both polysaccharides at high temperatures and is located to the cell surface, where it is directly involved in C. bescii attachment. Adherence to polysaccharides is likely key to survival in environments where carbon sources are limiting, allowing C. bescii to compete against other plant-degrading microorganisms.

Cell-associated and extracellular cellulolytic enzyme activity in the marine fungus Dendryphiella arenaria

1982 ◽  
Vol 60 (6) ◽  
pp. 838-844 ◽  
Author(s):  
Malcolm J. MacDonald ◽  
Marilyn K. Speedie
Keyword(s):  
Extracellular Enzymes ◽  
Marine Fungus ◽  
Crystalline Cellulose ◽  
Cellulolytic Enzyme ◽  
Cellulolytic Activity ◽  
Endoglucanase Activity ◽  
Produce Cell ◽  
Intracellular Fraction ◽  
Glucosidase Activity ◽  
Cell Fractions

Cellulolytic activity in Dendryphiella arenaria was demonstrated by its ability to release dye from dyed crystalline cellulose and to produce cell-associated and extracellular enzymes when grown on a similar substrate. Exoglucanase was found only in an insoluble form, associated with the hyphae and cellulose particles following sonic disruption of the cells. Endoglucanase was predominantly cell free but with a small amount associated with the cell fractions. High β-glucosidase activity was seen in the filtrate and cell fractions, although in the soluble intracellular fraction its activity disappeared with time. Optimal pH activities for exoglucanase and endoglucanase were demonstrated to be 7.0 and 5.8, respectively. Temperature optima of the enzyme components also differed; maximum exoglucanase activity in the cell-associated fractions and endoglucanase activity in the cell-associated and filtrate fractions occurred at 65 and 60 °C, respectively. Maximum β-glucosidase activity in the filtrate and soluble intracellular fractions occurred at 55 °C and that in the particulate fraction occurred at 60 °C.

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