Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.     

Screening for ethanol-producing filamentous fungi

Of nineteen Aspergilli and ten Rhizopus strains examined for their ability to ferment simple sugars (glucose, xylose, and arabinose) as well as complex substrates (cellulose, oat-spelt xylan, corn fiber, and corn germ pressing), three Rhizopus strains were identified that could produce more than 31 g ethanol/l under anaerobic stress. By 72 h, glucose , xylose, cellobiose, and corn fiber were fermented with perspective yields of 100, 47, 80, and 40 percent, of theoretical.     

Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.     

Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers.     

Product binding varies dramatically between processive and nonprocessive cellulase enzymes

Cellulases hydrolyze β-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.     

Pseudomonas fluorescens subsp. cellulosa: an alternative model for bacterial cellulase.

Pseudomonas fluorescens subsp. cellulosa, a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy. Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose. These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface. Cells of Ps. fluorescens subsp. cellulosa do not adhere to cellulose. In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose. Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps. fluorescens subsp. cellulosa and expressed in Escherichia coli, are synthesized by the pseudomonad under a variety of conditions. These enzymes appear to be post-translationally modified, probably through glycosylation. Overall, it appears that the cellulase/hemicellulase system of Ps. fluorescens subsp. cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum.     

Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Cellulase and Other Polymer-Hydrolyzing Activities of Trichomitopsis termopsidis, a Symbiotic Protozoan from Termites

Crude extracts of the anaerobic, cellulolytic protozoan Trichomitopsis termopsidis possessed endo-β-1,4-glucanase and cellobiase activities, as evidenced by hydrolytic action on carboxymethyl cellulose and cellobiose, respectively. Cell extracts also hydrolyzed microcrystalline cellulose. Hydrolysis of microcrystalline cellulose displayed optima at pH 5 and at 30°C, and glucose was the sole product liberated. Cellulolytic activities of T. termopsidis appeared to be entirely cell associated. Hydrolytic activity was also detected against Douglas fir wood powder, xylan, starch, and protein, but not chitin. The importance of these enzymes in the nutrition of T. termopsidis is discussed in terms of the natural habitat of this protozoan (the hindgut of wood-eating termites).     

Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.

Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly.     

The cellulase of Trichoderma viride. Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.     

Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.     

Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: A docking study

Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.     

High Speed Atomic Force Microscopy Visualizes Processive Movement of Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose

Fungal cellobiohydrolases act at liquid-solid interfaces. They have the ability to hydrolyze cellulose chains of a crystalline substrate because of their two-domain structure, i.e. cellulose-binding domain and catalytic domain, and unique active site architecture. However, the details of the action of the two domains on crystalline cellulose are still unclear. Here, we present real time observations of Trichoderma reesei (Tr) cellobiohydrolase I (Cel7A) molecules sliding on crystalline cellulose, obtained with a high speed atomic force microscope. The average velocity of the sliding movement on crystalline cellulose was 3.5 nm/s, and interestingly, the catalytic domain without the cellulose-binding domain moved with a velocity similar to that of the intact TrCel7A enzyme. However, no sliding of a catalytically inactive enzyme (mutant E212Q) or a variant lacking tryptophan at the entrance of the active site tunnel (mutant W40A) could be detected. This indicates that, besides the hydrolysis of glycosidic bonds, the loading of a cellulose chain into the active site tunnel is also essential for the enzyme movement.     

Water solubilization of DDGS via derivatization with phosphite esters

Ethanol production from corn starch in the corn dry milling process leaves Distillers' Dry Grains and Solubles (DDGS) as a major by-product from which additional ethanol may be economically obtained from its glucan content. A challenge in processing the cellulose content of this material lies in its extensive inter-cellulose chain hydrogen bonding, which inhibits access of enzymes capable of cleaving glycosidic bonds, a transformation required for providing fermentable sugars. The phosphitylation of cellulosic OH groups using a reactive bicyclic phosphite ester is utilized to disrupt cellulosic hydrogen bonds, thus providing access to cellulose chains for further processing. We describe a method of pretreating DDGS with commercially available trimethylolpropane phosphite [P(OCH2)3CEt] in the presence of a slight molar excess of water to afford greater than 90% DDGS solubility in the reaction mixture in methanol and in water. Preliminary results using a model compound [D-(+)-permethylated cellobiose] indicate that glycosidic bonds are cleaved as a consequence of this pretreatment.     

Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.