Reesei Cellulase Research Articles

In situ assay of the reducing sugars in hydrophilic natural deep eutectic solvents by a modified DNS method

The DNS assay has been already a universally method for the in situ determination of reducing sugars (RS) in aqueous phase solvents. Today, “greener biorefineries” require in situ measurement of reducing sugars in non-aqueous solvents such as natural deep eutectic solvents (NADESs). Therefore, that DNS assay cannot be performed successfully in such eutectic phase solvent becomes a puzzle that must be solved. In this work, an improved DNS method, which we call the DNS'' method, was established and implemented successfully in hydrophilic eutectic phase solvent systems (hiNADES). The tickets to the success of DNS'' method are the inserted two essential steps, water-adding disintegration and pH alkalization. Then we tested two excellent RS solvents (ChCl_Gly and ChCl_LA) from six synthetic ChCl-based hiNADESs. The optimal disintegration conditions for assaying RS in ChCl_LA/NaOH (ChCl_Gly/H2O) were volume ratio 3: 1 (3: 1), molar concentration 2.0 mol/L (0 mol/L). Thereafter, we drew the standard curves of seven RSs in ChCl_Gly and ChCl_LA, which are glucose, xylose, fructose, mannose, fucose, rhamnose, and maltose. We determined that HMF interferes with DNS assay in hiNADES. The principles and standard procedures of DNS'' method were also investigated. Finally, the enzymatic saccharification of microcrystalline cellulose (MCC) by two commercial Trichoderma reesei cellulases (Celluclast® 1.5 L and Cellic® CTec2) in neat hiNADES was tracked using the DNS'' method.

Improvements of the productivity and saccharification efficiency of the cellulolytic \u03b2-glucosidase D2-BGL in Pichia pastoris via directed evolution

Backgroundβ-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass.ResultsWe have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2.ConclusionsD2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

Alleviating product inhibition of Trichoderma reesei cellulase complex with a product-activated mushroom endoglucanase

Product inhibition of cellulase is a challenging issue in industrial processes. Here, we introduced a product-activated mushroom cellulase, PaCel3A from Polyporus arcularius, into Trichoderma reesei. The filter paper activity, carboxymethyl cellulase activity, and saccharification efficiency (substrate: pretreated rice straw, PRS) of transformants increased significantly with this enzyme (by 18.4–26.8%, 13.8–22.8%, and 17.0%, respectively). A mutant of PaCel3A, PaCel3AM, obtained based on B-factor analysis, saturated mutagenesis, and residual activity assay, showed improved thermostability. The PRS saccharification efficiency using the cellulase complex from T. reesei transformants overexpressing pacel3am increased by 56.4%–63.0%. In addition, the T. reesei cellulase complex obtained by adding the purified recombinant PaCel3AM from T. reesei (rCel3aM-tr) to hydrolyze PRS resulted in increased reducing sugar yields at all sampling points, outperforming the cellulase complexes without rCel3aM-tr. These results suggest that introducing product-activated cellulase genes is a simple and feasible method to alleviate the product inhibition of cellulase.

Sequential dilute acid and alkali deconstruction of sugarcane bagasse for improved hydrolysis: Insight from small angle neutron scattering (SANS)

Cellulosic bioethanol is a promising renewable and substitute source of energy. To make the bioconversion of LCB to fuels cost effective and energy efficient, it is essential to reduce the recalcitrance of LCB and unravel the process of biomass deconstruction. Present study employed sequential dilute acid-alkali pretreatment of sugarcane bagasse (SCB) for enhancing its bioconversion to ethanol. Box-Behnken and D-optimal designs were used to optimise the process of dilute acid and alkali pretreatments sequentially, resulting in an optimum concentration of 3% (v/v) and 5% (w/v) for H2SO4 and NaOH with solid SCB loadings of 18 and 15% (w/w), respectively, for 30 min at 121 °C. The effectiveness of sequential pretreatment was supported by increased cellulose content (83%), drop in hemicellulose, enhanced delignification and 60% enzymatic hydrolysis of SCB by in-house Trichoderma reesei cellulases at enzyme dose of 20 FPU/g. The favourable multi-length scale ultrastructural changes in SCB induced by pretreatment were confirmed by FT-IR, SEM, EDX, TGA, XRD and small angle neutron scattering (SANS). SANS revealed increase in small pore radii from 11.1 to 18.5 Å, indicating improved biomass porosity after sequential pretreatment. Thus, sequential pretreatment of LCB effectively reduced the recalcitrance and could be more useful in lignocellulosic biorefinery applications.

CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei.

The stringent regulatory network of cellulase gene expression in the filamentous fungus Trichoderma reesei involves multiple transcriptional regulators. However, identification and mechanistic investigation of these regulators are still insufficient. Here, we identified a novel transcriptional regulator, CLP1, a plant homeo domain (PHD) Protein that participates in regulating T. reesei cellulase gene expression. Phylogenetic analyses demonstrated that CLP1 homologs are widely distributed in filamentous fungi including Trichoderma, Penicillium, Fusarium, Neurospora, and Aspergillus species. We demonstrated that CLP1 is a nuclear protein and lack of CLP1 significantly impaired the induced expression of cellulase genes. ChIP experiments showed CLP1 binding to the cellulase gene promoters specifically under cellulose conditions and compromised XYR1 occupancy on the same promoters in the absence of CLP1 at the early induction stage. XYR1 overexpression fully rescued the defect in cellulase production but not the defect in conidia formation in the clp1 null mutant. Further analysis showed that the PHD is required for the CLP1 appropriate subcellular localization as well as the induced cellulase gene expression and conidiation. Taken together, these data demonstrated an important role of CLP1 in the regulation of cellulase and xylanase gene expression in T. reesei.

Heterologous expression of Neurospora crassa cbh1 gene in Pichia pastoris resulted in production of a neutral cellobiohydrolase I.

The high production cost of cellulase is one of the limitations that hinder the commercialization of lignocellulose-based biorefineries. As one of the important cellulases, Neurospora crassa cellulase is not so intensively investigated as T. reesei cellulase. In this study, the cbh1gene (NCU07340) cloned from N. crassa was expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promotor. Six transformants with the highest resistance to G418 were selected by two rounds of transformant screening, among which the most robust producer of the recombinant cellobiohydrolase I (CBHI) has an Avicelase activity of 0.22 U/mL. After fermentation optimization, it was improved to 0.30 U/mL. Interestingly, the optimal temperature and pH of the recombinant CBHI were 60°C and 7.2, respectively, and it was quite stable within the wide ranges of temperature and pH. This work is a good example for the future improvement and optimization of N. crassa cellulase.

Direct ethanol production from cellulose by consortium of Trichoderma reesei and Candida molischiana

Abstract Industrial cellulosic ethanol production is a challenge due to the high cost of cellulases for hydrolysis when lignocellulosic materials are used as feedstock. In this study, direct ethanol production from cellulose was performed by consortium of Trichoderma reesei and Candida molischiana. Cellulose was hydrolyzed by a fully enzymatic saccharification process using Trichoderma reesei cellulases. The produced reducing sugar was further utilized by Candida molischiana for ethanol production. Because the optimal temperature for the cellulase system is approximately 50°C, the effect of temperature rise from 30°C to 50°C on cellulose hydrolysis was investigated. The results showed that the temperature rise from 30°C to 50°C after 36 h of cultivation was the best for reducing sugar and glucose production. Under these conditions, the maximum concentrations of reducing sugar and glucose produced by T. reesei were 8.0 g/L and 4.6 g/L at 60 h, respectively. The maximum production of ethanol by C. molischiana was 3.0 g/L after 120 h.

A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting

BackgroundIn the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive.ResultsHerein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library.ConclusionsThe coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65°C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

Bioethanol production from rice husk using different pretreatments and fermentation conditions.

Bioethanol is an environmentally friendly alternative to petroleum energy sources. This study evaluated the effects of H2O, HCl, NaOH and FeCl3 pretreated rice husk feedstocks on the production of bioethanol. The pretreatments were carried out using water, 0.1M HCl, NaOH and FeCl3 at 121°C for 15min, followed by simultaneous saccharification and fermentation (SSF) as well as separate hydrolysis and fermentation (SHF). The raw and pretreated lignocellulosic feedstocks were analyzed using Fourier transform infrared spectroscopy. Saccharification and fermentation were accomplished using Trichoderma reesei cellulase and Saccharomyces cerevisiae, respectively. The products obtained after saccharification and fermentation were collected and analyzed for reducing sugars and ethanol contents using 3,5-dinitrosalicylic acid and high-performance liquid chromatography, respectively. Enzyme hydrolysis of the FeCl3 and HCl treated samples resulted in hydrolysates containing 3.845 and 3.402mg/ml glucose equivalent, respectively. In all pretreatments, SSF for each pretreatment produced more ethanol than the SHF method; the FeCl3 pretreatment gave the highest ethanol yield of 3.011±0.034 and 3.802±0.041% in the SHF and SSF methods, respectively. Utilization of FeCl3 pretreatment of rice husk is a potential option for bioethanol production in the future.

Improved thermostability of a metagenomic glucose-tolerant β-glycosidase based on its X-ray crystal structure.

MeBglD2, a metagenomic β-glycosidase, is stimulated by various saccharides, including D-glucose, D-xylose, and maltose, and it promotes the enzymatic saccharification of plant biomass. To improve the thermostability of MeBglD2, its X-ray crystal structure was analyzed, and the amino acid residues responsible for its thermostability were identified using the structural information. Mutations in His8, Asn59, and Gly295 improved the thermostability of MeBglD2, and the combination of these mutations resulted in the highest thermostability. Compared with wild-type MeBglD2, thermostable MeBglD2 mutants promoted plant biomass saccharification using Trichoderma reesei cellulase. In addition to thermostability, the thermostable mutants exhibited higher tolerance to ethanol, dimethyl sulfoxide, and copper ions, indicating that the MeBglD2 mutants generated in this study were improved in their tolerance to not only high temperature but also to organic solvents and metal ions.

Quantitative Effects of O-Linked Glycans on Protein Folding.

Protein O-glycosylation is a diverse, common, and important post-translational modification of both proteins inside the cell and those that are secreted or membrane-bound. Much work has shown that O-glycosylation can alter the structure, function, and physical properties of the proteins to which it is attached. One gap remaining in our understanding of O-glycoproteins is how O-glycans might affect the folding of proteins. Here, we took advantage of synthetic, homogeneous O-glycopeptides to show that certain glycosylation patterns have an intrinsic effect, independent of any cellular folding machinery, on the folding pathway of a model O-glycoprotein, a carbohydrate binding module (CBM) derived from the Trichoderma reesei cellulase TrCel7A. The strongest effect, a 6-fold increase in overall folding rate, was observed when a single O-mannose was the glycan, and the glycosylation site was near the N-terminus of the peptide sequence. We were also able to show that glycosylation patterns affected the kinetics of each step in unique ways, which may help to explain the observations made here. This work is a first step toward quantitative understanding of how O-glycosylation might control, through intrinsic means, the folding of O-glycoproteins. Such an understanding is expected to facilitate future investigations into the effects of glycosylation on more biological processes related to protein folding.

Genetic engineering of Trichoderma reesei cellulases and their production.

SummaryLignocellulosic biomass, which mainly consists of cellulose, hemicellulose and lignin, is the most abundant renewable source for production of biofuel and biorefinery products. The industrial use of plant biomass involves mechanical milling or chipping, followed by chemical or physicochemical pretreatment steps to make the material more susceptible to enzymatic hydrolysis. Thereby the cost of enzyme production still presents the major bottleneck, mostly because some of the produced enzymes have low catalytic activity under industrial conditions and/or because the rate of hydrolysis of some enzymes in the secreted enzyme mixture is limiting. Almost all of the lignocellulolytic enzyme cocktails needed for the hydrolysis step are produced by fermentation of the ascomycete Trichoderma reesei (Hypocreales). For this reason, the structure and mechanism of the enzymes involved, the regulation of their expression and the pathways of their formation and secretion have been investigated in T. reesei in considerable details. Several of the findings thereby obtained have been used to improve the formation of the T. reesei cellulases and their properties. In this article, we will review the achievements that have already been made and also show promising fields for further progress.

Characterization of endoglucanase rich Trichoderma reesei cellulase mixtures and their effect on alkaline solubility of dissolving pulp

Dissolving grade pulps are used to manufacture regenerated cellulosic fibres. One promising process for the production of regenerated fibres utilises endoglucanse rich cellulases in the modification of dissolving pulp into alkaline soluble form. The aim of this paper was to characterise cellulases produced by Trichoderma reesei that are available in large quantities and study their effect on the dissolving grade softwood pulp, especially on its alkaline solubility. All the studied cellulases had endoglucanse activity and they decreased the intrinsic viscosity of the pulp. The degradation of cellulose into solubilised sugars increased with the cellulases containing also cellobiohydrolases. The monocomponent endoglucanases enhanced alkaline solubility of the pulp more than the multicomponent cellulases and produced alkaline solutions with higher fluidity. The studies showed that the type of the cellulases in the enzyme mixture has significant effect on the amount of solubilised sugars during the enzyme treatment and on the alkaline solubility of the pulp.

Screening, identification, and characterization of a novel saccharide-stimulated β-glycosidase from a soil metagenomic library.

MeBglD2, a β-glycosidase that is highly activated in the presence of various monosaccharides and disaccharides, was isolated from a soil metagenomic library. MeBglD2 had not only β-glucosidase activity but also β-galactosidase and β-fucosidase activities. MeBglD2 β-glucosidase activity increased in a cellobiose concentration-dependent manner and was not inhibited by a high concentration of D-glucose or cellobiose. MeBglD2 β-glucosidase and β-fucosidase activities were activated by various monosaccharides and disaccharides including D-glucose, D-xylose, D-galactose, maltose, and cellobiose. The saccharification yield of rice straw using Trichoderma reesei cellulase was improved by the addition of MeBglD2. These results show that MeBglD2 can be used to improve plant biomass saccharification, because both substrates and products can activate its enzymatic activity.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities

BackgroundThe filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently.ResultsThe thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3–69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose.ConclusionsWe report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

Production of nanotubes in delignified porous cellulosic materials after hydrolysis with cellulase

In this study, tubular cellulose (TC), a porous cellulosic material produced by delignification of sawdust, was treated with a Trichoderma reesei cellulase in order to increase the proportion of nano-tubes. The effect of enzyme concentration and treatment duration on surface characteristics was studied and the samples were analyzed with BET, SEM and XRD. Also, a composite material of gelatinized starch and TC underwent enzymatic treatment in combination with amylase (320U) and cellulase (320U) enzymes. For TC, the optimum enzyme concentration (640U) led to significant increase of TC specific surface area and pore volume along with the reduction of pore diameter. It was also shown that the enzymatic treatment did not result to a significant change of cellulose crystallinity index. The produced nano-tubular cellulose shows potential for application to drug and chemical preservative delivery systems.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover.

BackgroundNon-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.ResultsIn this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration.ConclusionWe propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation

Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173gglc/FPU, higher than the binary ACR:XYL mixture (0.122gglc/FPU) and ACR alone (0.081gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry.

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed β-glucosidase had a positie effect on cell growth in agreement with the fact that no putative β-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.