Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.     

Evaluation of cellulases produced from four fungi cultured on furfural residues and microcrystalline cellulose

Four fungal strains—Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei—were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase, endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic materials in ethanol production.     

Saccharification of used paper with different cellulases

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.     

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

     

Induction and production of cellulases by l-sorbose in Trichoderma reesei

Summary Cellulase production in Trichoderma reesei mutants was induced by l-sorbose, known to be an inhibitor of -1,3-glucan synthesis. In the experiments the washed mycelia were used as resting cells. For CMCase induction over 24 h using T. reesei PC-3-7, the most effective pH, temperature and l-sorbose concentration were 2.8, 28° C and 0.3 mg/ml, respectively. Comparison with other cellulase inducers showed that the inductive level of CMCase by l-sorbose was similar to that by sophorose, known to be the most potent inducer of cellulases. Since the induction of CMCase was inhibited completely by 10 g of cycloheximide per ml, the induction process was considered to involve de novo synthesis. Although l-sorbose had the effective inducibility of CMCase, the assimilation rate of l-sorbose was very low in T. reesei PC-3-7.Production of Ethanol from Biomasses. Part III.Production of Ethanol from Biomasses. Part III.     

Heterologous expression of <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> cellobiohydrolase Cel7A in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> increases the efficiency of corncob residues saccharification

Objective

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii.

Results

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.

Conclusions

The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

     

Cellulase production by Trichoderma reesei immobilized on κ-carrageenan

Summary Cellulase production by Trichoderma reesei mutant RUT-C30, immobilized on 4% -carrageenan beads, was monitored in continuous culture for 13 days. Cellulase production averaged 26.0 Filter Paper Units (FPU)/l/hr; carbon and nitrogen requirements per FPU produced were reduced to 1/4-1/2 those of conventional continuous culture.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

ATP measurement for cellulase production control

Summary Plotting intracellular ATP concentrations as a function of time during cellulase production byTrichoderma reesei, provides a good evaluation of fungal growth and specific rate of cellulose consumption. ATP content evaluation is of great help for running fed-batch and semi-continuous fermentations.     

Construction of a promoter collection for genes co-expression in filamentous fungus Trichoderma reesei

Trichoderma reesei is the preferred organism for producing industrial cellulases. However, cellulases derived from T. reesei have their highest activity at acidic pH. When the pH value increased above 7, the enzyme activities almost disappeared, thereby limiting the application of fungal cellulases under neutral or alkaline conditions. A lot of heterologous alkaline cellulases have been successfully expressed in T. reesei to improve its cellulolytic profile. To our knowledge, there are few reports describing the co-expression of two or more heterologous cellulases in T. reesei. We designed and constructed a promoter collection for gene expression and co-expression in T. reesei. Taking alkaline cellulase as a reporter gene, we assessed our promoters with strengths ranging from 4 to 106 % as compared to the pWEF31 expression vector (Lv D, Wang W, Wei D (2012) Construction of two vectors for gene expression in Trichoderma reesei. Plasmid 67(1):67–71). The promoter collection was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. We observed higher activities of both cellulose degradation and biostoning by the co-expression of an endoglucanase and a cellobiohydrolase than the activities obtained by the expression of only endoglucanase or cellobiohydrolase. This study makes the process of engineering expression of multiple genes easier in T. reesei.     

Use of pretreated lignocellulose for the production of cellulases

Summary Tests made to utilize lignocellulosics as a substrate for the production of cellulases showed that the enzyme production from steam and explosion decompressed aspen wood (SED) by Tricoderma reesei RUT-C30 was low, and the enzyme system produced was deficient in exoglucanase and -glucosidase activities. Mixing this substrate with 10–20% pure cellulose lessened this deficiency and improved enzyme production. The enzyme system produced from the mixed substrate was rich in xylanase and had saccharifying ability equal to that produced in medium containing pure cellulose.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Induction of cellulase formation in Trichoderma reesei by cellobiono-1,5-lacton

Induction of synthesis of cellulolytic enzymes in Trichoderma reesei QM 9414 by cellobiono-1,5-lactone (CBL) has been investigated in a replacement system lacking additional carbon source. CBL induced cellulase secretion optimally at pH 5 and a concentration of 70 g/ml. Higher concentrations lead to lower induction. De novo induction of cellulases was proven by the inhibitory effect of cycloheximide addition. Induction by CBL was shown to act synergistically on induction by sophorose, as it decreased the concentration of sophorose required for maximal induction. Maximal endo--1,4-glucanase activities induced by either sophorose or CBL were comparable. The CBL-induced cellulase system contained all the major cellulolytic enzymes of T. reesei, i.e. cellobiohydrolase I and II, and endoglucanase I, as shown by SDS-PAGE, Western blotting and detection with specific mono- and polyclonal antibodies. No differences were seen in the types of individual enzymes formed upon induction by either sophorose or CBL. No other hydrolytic enzymes appear to be induced by CBL (i.e. amylase, laminarinase, xylanase).Abbreviations SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium-dodecylsulfate - CBL cellobiono-1,5-lacton - CBH cellobiohydrolase - EG endoglucanase - IgG immunoglobulin G