Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.     

Isolation and purification of isoenzymes of cellobiohydrolase I and II of trichoderma reesei using LPLC methods

Five cellulases were fractionated from a commercial cellulase preparation (Celluclast^TM) Two isoenzymes of cellobiohydrolase I (CBHI)(pI = 4.1) could be proved to be real exo-glucanases due to their activity towards MU (=methylumbelliferyl)-lactoside being inhibited by cellobiose (5 mM) and due to production of cellobiose from carboxymethylcellulose (CMC) as the sole final product.Two isoenzymes of CBHII (pI=6.15, 6.0) were shown to act as endo-glucanases because they produced glucose, cellobiose and cellotetraose from CMC and because they were not inhibited by cellobiose when decomposing MU-lactoside. Results confirm recent reports in the literature classifying CBHI and CBHII as exo-type and endo-type cellulases, respectively. Both the CBHI and the CBHII isoenzymes were shown to be active towards CMC and amorphous cellulose.CBHI and CBHII reactions could be differentiated from one another by the velocities of decomposition of CMC: CBHI acts slowly and linearly whereas CBHII acts strongly and exponentially.The fifth of the purified enzymes must be classed as a conventional endoglucanase which exhibits activity towards CMC but fails to be active towards MU-lactoside and amorphous cellulose.     

Improvement of cellulose-degrading ability of a yeast strain displaying Trichoderma reesei endoglucanase II by recombination of cellulose-binding domains

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to 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.     

The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size

Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.     

Functional analysis of a gene encoding endoglucanase that belongs to glycosyl hydrolase family 12 from the brown-rot basidiomycete Fomitopsis palustris

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and beta- glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.     

Adsorption of major endoglucanase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> on cellulosic substrates

A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated A _max and K _ad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing.     

In vitro assembly of minicellulosomes with two scaffoldins on the yeast cell surface for cellulose saccharification and bioethanol production

The present paper reports in vitro strategies for assembly of minicellulosomes with two miniscaffoldins on the Saccharomyces cerevisiae cell surface. It was carried out through incubation of the yeast cells displaying scaffoldins with Escherichia coli lysates containing recombinant cellulases, or using a four-population yeast consortium. The results showed that the display level of miniscaffoldin II was distinctly increased by moving the cellulases production into E. coli or other yeast cells, indicating that the metabolic burden of the yeast host was decreased. The yeast consortium did not show any cellulolytic activity, while the E. coli lysates-treated yeast, whose anchoring miniscaffoldin length was optimized, was able to produce ～1138 mg/L ethanol from microcrystalline cellulose within 4 days. We also confirmed that the yeast-associated minicellulosome moreover showed both higher thermal stability and lower protease accessibility than free minicellulosome. This research promotes the application of S. cerevisiae as a consolidated bioprocessing (CBP) microorganism in cellulosic bioethanol production.     

Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity

Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via δ-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 ± 0.51, 18.60 ± 0.81, 28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi.     

Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced lichenan utilization and solvent production.

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.     

Degradation of crystalline cellulose by the brown-rot basidiomycete Fomitopsis palustris

This study demonstrated that the brown rot basidiomycete Fomitopsis palustris was able to degrade crystalline cellulose (Avicel). This fungus could also produce the three major cellulases (exoglucanases, endoglucanases, and beta-glucosidase) when the cells were grown on 2.0% Avicel. Avicel degraded by F. palustris showed a decrease in relative crystallinity from 83% to 78.5% after 14 days of incubation. The characterization study indicated that optimum pH was 4.5 and optimum temperature was 70 degrees C for exoglucanase (cellobiohydrolase) activity. Hydrolysis of Avicel by the crude enzyme from F. palustris yielded 1.6 mg/ml of glucose after 43 h, which corresponded to a cellulose conversion degree of 3.2%. Therefore, this study revealed for the first time that the brown rot basidiomycete F. palustris produces cellulases capable of yielding soluble sugars from crystalline cellulose.     

Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface

Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.     

Screening of optimal cellulases from symbiotic protists of termites through expression in the secretory pathway of Saccharomyces cerevisiae

For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.     

Extremely thermophilic microorganisms for biomass conversion: status and prospects

Many microorganisms that grow at elevated temperatures are able to utilize a variety of carbohydrates pertinent to the conversion of lignocellulosic biomass to bioenergy. The range of substrates utilized depends on growth temperature optimum and biotope. Hyperthermophilic marine archaea (T(opt)>or=80 degrees C) utilize alpha- and beta-linked glucans, such as starch, barley glucan, laminarin, and chitin, while hyperthermophilic marine bacteria (T(opt)>or=80 degrees C) utilize the same glucans as well as hemicellulose, such as xylans and mannans. However, none of these organisms are able to efficiently utilize crystalline cellulose. Among the thermophiles, this ability is limited to a few terrestrial bacteria with upper temperature limits for growth near 75 degrees C. Deconstruction of crystalline cellulose by these extreme thermophiles is achieved by 'free' primary cellulases, which are distinct from those typically associated with large multi-enzyme complexes known as cellulosomes. These primary cellulases also differ from the endoglucanases (referred to here as 'secondary cellulases') reported from marine hyperthermophiles that show only weak activity toward cellulose. Many extremely thermophilic enzymes implicated in the deconstruction of lignocellulose can be identified in genome sequences, and many more promising biocatalysts probably remain annotated as 'hypothetical proteins'. Characterization of these enzymes will require intensive effort but is likely to generate new opportunities for the use of renewable resources as biofuels.     

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.     

Synergism of Trichoderma reesei cellulases while degrading different celluloses

Summary The synergistic action of purified cellulases from Trichoderma reesei in hydrolysis of cellulose decreased with increasing substrate concentration, depended strongly on the the type of cellulose used, and was maximal on crystalline cellulose. Contrarily, the activity of the individual cellulases was highest on amorphous cellulose. The binary combinations CBH I/EG III and CBH I/CBH II exhibited the greatest degree of synergism on crystalline cellulose.     

Screening of Optimal Cellulases from Symbiotic Protists of Termites through Expression in the Secretory Pathway of Saccharomyces cerevisiae

For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.     

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.