Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β-Glucosidase BGLII (Cel1A)

This paper describes the characterization of an intracellular β-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific β-glucosidase, having no β-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this β-glucosidase.     

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

Background  

The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.     

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.     

Management of palm oil mill effluent through production of cellulases by filamentous fungi

A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to evaluate the bioconversion process.     

Spent brewery grains as substrate for the production of cellulases byTrichoderma reesei QM9414

Summary The cellulolytic fungusTrichoderma reesei QM9414 can be cultivated on spent brewery grains for the production of cellulases. The levels of the cellulase components endoglucanase and exoglucanase synthesized, and the complexes filter paper cellulase and grain-hydrolyzing cellulase synthesized by the organism on spent grains were as high as 287, 182, 187, and 449 units per g available cellulose, respectively. Scaling up the spent grains fermentation system by up to 40-fold (200g dry substrate/tray) demonstrated that cellulase production was comparable to laboratory scale (5g dry substrate/flask) yields. Cultivation of the fungus was feasible on spent grains without pretreatment or further adjustment, although the enzyme yield was somewhat lower than that on dried grains moistened with water orTrichoderma medium. This suggested the possible reutilization of spent grains, with minimal pretreatment, in the cultivation ofT.reesei QM9414 for cellulase synthesis and for future incorporation into animal feed.     

A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes

Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a P _tcu1 -based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the P _tcu1 -ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.

     

The production of highly effective enzyme complexes of cellulases and hemicellulases based on the Penicillium verruculosum strain for the hydrolysis of plant raw materials

Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively.     

Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Agrobacterium-mediated T-DNA transfer has been proven to be an efficient strategy for insertional mutagenesis and elucidation of gene function in filamentous fungi. The implementation of large-scale T-DNA insertional mutagenesis requires the development of high-efficient transformation and high-throughput screening procedures. Here, using green fluorescent protein (GFP) as a vital marker, a highly efficient T-DNA-based mutagenesis and screening system was developed in Trichoderma reesei. The uridine auxotrophic T. reesei M23 as the host was transformed with A. tumefaciens EH105 strain harboring a binary vector pC-OEP, which beared the pyrG gene for primary selection on minimal medium without uridine and the egfp gene for fluorescence-based rapid screening of the mitotically stable transformants. The efficiency of transformation was up to 10–20 transformants per 10⁵ target conidia. Microscopic examination revealed strong GFP expression and fluorescence emission in conidia, growing hyphae and mycelia. An effective and convenient screening procedure using 96-well plates and multilabel counter for fluorescence intensity counting was developed to rapidly identify the T-DNA tagged T. reesei mutants. Furthermore, the presence of T-DNA integration at random sites in the genome was confirmed by Southern blot analysis. This report of the T-DNA-based mutagenesis and rapid screening system using GFP as a vital reporter provides a promising strategy to speeding up the genome-scale T-DNA insertional mutagenesis and functional genomics analysis of this cellulolytic fungus T. reesei.     

The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form

Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C₂H₂ type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.     

Identification of microRNA-Like RNAs in the Filamentous Fungus Trichoderma reesei by Solexa Sequencing

microRNAs (miRNAs) are non-coding small RNAs (sRNAs) capable of negatively regulating gene expression. Recently, microRNA-like small RNAs (milRNAs) were discovered in several filamentous fungi but not yet in Trichoderma reesei, an industrial filamentous fungus that can secrete abundant hydrolases. To explore the presence of milRNA in T. reesei and evaluate their expression under induction of cellulose, two T. reesei sRNA libraries of cellulose induction (IN) and non-induction (CON) were generated and sequenced using Solexa sequencing technology. A total of 726 and 631 sRNAs were obtained from the IN and CON samples, respectively. Global expression analysis showed an extensively differential expression of sRNAs in T. reesei under the two conditions. Thirteen predicted milRNAs were identified in T. reesei based on the short hairpin structure analysis. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. Computational analysis predicted a number of potential targets relating to many processes including regulation of enzyme expression. The presence and differential expression of T. reesei milRNAs imply that milRNA might play a role in T. reesei growth and cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

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.     

Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens

Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I (cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation.     

Effect of pH and temperature on the global compactness, structure, and activity of cellobiohydrolase Cel7A from Trichoderma harzianum

Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum (T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A from T. harzianum (Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity of Th Cel7A at different pH and temperature conditions.