Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β-d-glucosidase (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β-d-glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. d-Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. d-Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while d-fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β-d-glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

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.     

Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1.     

New chromosome counts in nine endemic species from Iran

Original meiotic chromosome counts are presented for nine endemic species in seven families of Angiosperms from Iran including:Arum giganteum Ghahr. (Araceae) (n=14),Caccinia actinobole Bunge (Boraginaceae) (n=8),Delphinium aquilegifolium (Boiss.)Bornm. (Ranunculaceae) (n=8),Diplotaenia damavandica Mozaff., Hedge etLamond (Apiaceae) (n=11),Gypsophila caricifolia Boiss. (Caryophyllaceae) (n=17),Iphiona arachnoidea (Boiss.)Anderb. (Asteraceae) (n=9),Moltkia gypsacea Rech. f. etAellen (Boraginaceae) (n=20),Onobrychis gaubae Bornm. (Fabaceae) (n=8) andOnosma platyphyllum Riedl (Boraginaceae) (n=9). Eight counts are reported for the first time. Furthermore, the previous chromosome count forIphiona aracnoidea is corrected. Based on cytological data the species status ofMoltkia gypsacea is confirmed; it is not merely synonymous withM. coerulea (Willd.)Lehm. The basic chromosome number n=11 is reported in the genusDiplotaenia for the first time.     

Contrasting soil fungal community responses to experimental nitrogen addition using the large subunit rRNA taxonomic marker and cellobiohydrolase I functional marker

Human activities have resulted in increased nitrogen inputs into terrestrial ecosystems, but the impact of nitrogen on ecosystem function, such as nutrient cycling, will depend at least in part on the response of soil fungal communities. We examined the response of soil fungi to experimental nitrogen addition in a loblolly pine forest (North Carolina, USA) using a taxonomic marker (large subunit rDNA, LSU) and a functional marker involved in a critical step of cellulose degradation (cellobiohydrolase, cbhI) at five time points that spanned fourteen months. Sampling date had no impact on fungal community richness or composition for either gene. Based on the LSU, nitrogen addition led to increased fungal community richness, reduced relative abundance of fungi in the phylum Basidiomycota and altered community composition; however, similar shifts were not observed with cbhI. Fungal community dissimilarity of the LSU and cbhI genes was significantly correlated in the ambient plots, but not in nitrogen‐amended plots, suggesting either functional redundancy of fungi with the cbhI gene or shifts in other functional groups in response to nitrogen addition. To determine whether sequence similarity of cbhI could be predicted based on taxonomic relatedness of fungi, we conducted a phylogenetic analysis of publically available cbhI sequences from known isolates and found that for a subset of isolates, similar cbhI genes were found within distantly related fungal taxa. Together, these findings suggest that taxonomic shifts in the total fungal community do not necessarily result in changes in the functional diversity of fungi.     

Cloning, Heterologous Expression, and Characterization of the Xylitol and l-Arabitol Dehydrogenase Genes, Texdh and Telad, from the Thermophilic Fungus Talaromyces emersonii

The genes encoding xylitol dehydrogenase (Texdh) and l-arabitol dehydrogenase (Telad) are involved in the fungal pentose pathway and were isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli, and the products purified to homogeneity. TeXDH showed activity toward xylitol and d-sorbitol. TeLAD was active with l-arabitol, xylitol, and d-sorbitol. Phylogenetic analysis showed TeLAD has evolved from d-sorbitol dehydrogenase as a result of environmental adaptation. Substrate specificity studies indicate that TeXDH is likely to have evolved from the more broadly acting TeLAD. Texdh and Telad expression was inducible by the same carbon sources responsible for induction of genes involved in biomass degradation, suggesting for the first time a coordinated regulatory control mechanism for expression of genes encoding extracellular hydrolases and intracellular metabolic genes in the pentose utilization pathways of T. emersonii. These data also suggest that TeXDH and TeLAD may be valuable in the production of xylitol, l-arabitol, and ethanol from renewable resources rich in pentose sugars.     

Cellulose-inducible Ultrastructural Protuberances and Cellulose-affinity Proteins of Eubacterium cellulosolvens

Scanning electron microscopy detected ultrastructural protuberances on the cellulolytic anaerobeEubacterium cellulosolvens . Such cell surface structures were found only when cells were cultivated in cellulose containing medium, suggesting these structures play a role in cellulose degradation. Organisms cultivated in medium containing cellobiose, glucose, fructose, maltose, or carboxymethylcellulose (CMC) contained few, if any, of these protuberances. Also, when a soluble carbohydrate or CMC was added to cellulose-grown cells, the ultrastructural protuberances were no longer detected. In fact, a time course study revealed that the loss of these protuberant structures occurred within 5 min of the addition of glucose, cellobiose, fructose, or a glucose analog to the medium. On the other hand, formation of these protuberances required at least 2 h, and 4 h before large numbers were present on the cells. Cellulose-grown cells also bound the FITC-labeled lectin BSI-B₄, obtained from Bandeiraea (formerly Griffonia) simplicifolia. Less detectable levels of lectin were bound by cellobiose-grown cells, and glucose- and fructose-grown cells did not bind any detectable levels of the lectin. Moreover, the addition of glucose or 2-deoxyglucose to the medium of a cellulose-grown culture resulted in the loss of detectable lectin binding. A cellulose-affinity protein fraction, which contained cellulase activity, was also isolated from the cellular extracts of cellobiose- and cellulose-grown cultures of E. cellulosolvens. This affinity fraction could not be eluted from the cellulose column with either sodium dodecyl sulfate (SDS), urea, or a 2-M solution of NaCl, but was eluted by Tris buffer containing ethylenediaminetetraacetic acid (EDTA). The fraction possessed cellulase activity, and consisted of numerous polypeptides. However, this protein fraction could not be detected in the extract of glucose-grown cultures, or in the extract of cellulose-grown cultures within 5 min of the addition of glucose (or a glucose analog) to the medium. The immediate loss of the cellulose-affinity protein fraction and protuberant structures when a soluble carbohydrate was added to the medium indicated some, as yet unknown, regulatory mechanism.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Bioethanol production by heterologous expression of Pdc and AdhII in Streptomyces lividans

Two genes from Zymomonas mobilis that are responsible for ethanol production, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII), were heterologously expressed in the Gram-positive bacterium Streptomyces lividans TK24. An examination of carbon distribution revealed that a significant portion of carbon metabolism was switched from biomass and organic acid biosynthesis to ethanol production upon the expression of pdc and adhII. The recombinant S. lividans TK24 produced ethanol from glucose with a yield of 23.7 % based on the carbohydrate consumed. The recombinant was able to produce ethanol from xylose, l-arabinose, mannose, l-rhamnose, galactose, ribose, and cellobiose with yields of 16.0, 25.6, 21.5, 33.6, 30.6, 14.6, and 33.3 %, respectively. Polymeric substances such as starch and xylan were directly converted to ethanol by the recombinant with ethanol yields of 18.9 and 8.8 %, respectively. The recombinant S. lividans TK24/Tpet developed in this study is potentially a useful microbial resource for ethanol production from various sources of biomasses, especially microalgae.     

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V_max values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K_m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).     

Fluxes and compartmentation of 3-O-methyl-D-glucose in Riccia fluitans

In thalli of the aquatic liverwort, Riccia fluitans, 3-O-methyl-D-glucose (3-OMG) is not metabolized. Intracellular compartmentation, accumulation and transmembrane fluxes of 3-OMG have been determined by compartmental analysis. A novel set of equations has been derived to extend this method to non-steady-state conditions of constant but unequal unidirectional fluxes. Efflux kinetics with 3-OMG and L-glucose revealed two intracellular flux compartments, presumably cytoplasm and vacuole; an additional quickly exchanging compartment (half-time approx. 1 min) has been assigned to the apoplast. With 1 mM 3-OMG given externally, cytoplasmic 3-OMG concentration (c _c) attains a quasi-steady state of about 10 mM lasting for >100 h, whereas the presumed vacuolar concentration (c _v) rises steadily, but does not reach flux equilibrium even after two weeks (c _v=46 mM). After 24 h incubation with 0.03 mM 3-OMG, c _c=1 mM approx., and c _v=3 mM approx., thus indicating accumulation by active hexose transport at both the plasmalemma and tonoplast. External D-glucose, but no D-mannitol, competitively inhibits 3-OMG uptake (cis-inhibition) and stimulates 3-OMG efflux at the plasmalemma by a factor up to 2.5. This trans-stimulation saturates half-maximally at 1.5 mM D-glucose. It clearly indicates a hexose carrier in the plasmalemma with one substrate-binding site for D-glocose and 3-OMG, alternately exposed to the cytoplasmic and outside compartment. The extent of the measured trans-stimulation can only be explained, if in the transport cycle the translocation of the empty substrate-binding site across the plasmalemma is rate-limiting.     

Interrelationship of Xylanase Induction and Cellulase Induction of Trichoderma longibrachiatum

Xylose oligomers rapidly induced xylanase activity of Trichoderma longibrachiatum, whereas induction was delayed in the presence of glucose. Cellobiose, cellopentaose, and xylobiose did not induce detectable levels of cellulase activity. However, mixtures of xylobiose with cellobiose or cellopentaose rapidly induced cellulase activity. In addition, mixtures of xylobiose with cellopentaose or cellobiose induced xylanase activity more effectively than xylobiose alone. Both xylanase and cellulase activity were detected after a lag period in the presence of lactose.