Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei,the hyper-cellulolytic filamentous fungus

The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.     

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.     

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.     

Flow cytometric sorting of the filamentous fungus Trichoderma reesei for improved strains

Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I (cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass.     

Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei

Journal of Industrial Microbiology & Biotechnology - The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial...     

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity.     

Determining the environmental fate of a filamentous fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating

Trichoderma spp. are used extensively in industry and are routinely disposed of in landfill sites as spent biomass from fermentation plants. However, little is known regarding the environmental fate of this biomass. We tracked the survival of T. reesei strain QM6A#4 (a derivative of strain QM6A marked with a recombinant construct) over a 6-month period in laboratory-contained, intact soil-core microcosms incubated in a growth chamber. Survival was tested in 3 different soils and the effect of a plant rhizosphere (bush lima beans, Phaseolus limensis) was investigated. Levels and viability of the fungus were determined, respectively, by quantitative competitive polymerase chain reaction analysis of total soil DNA extracts and dilution-plating of soil on a semiselective growth medium. Whereas chemically killed QM6A#4 became undetectable within 3 d, QM6A#4 added as a live inoculum decreased approximately 4- to approximately 160-fold over the first 1-3 months and then reached a steady state. After 4 months, soil cores were subjected to a 1.5-month simulated winter period, which did not significantly affect QM6A#4 levels. Throughout the experiment, QM6A#4 remained viable. These results indicate that, following release into the environment, live T. reesei will persist in soil for at least 2 seasons.     

A complete protein pattern of cellulase and hemicellulase genes in the filamentous fungus Trichoderma reesei

The complete protein pattern of cellulase and hemicellulase genes was studied through the Genome-wide analysis in Trichoderma reesei. The genome database revealed the presence of 39 ORFs encoding related proteins, including 32 enzymes with a catalysis domain related to cellulases and hemicellulases and 7 related proteins with a cellulose-binding module (CBM). Ten of these encoded yet undescribed enzymes, including six novel beta-glucosidases or xylosidases, two putative xylanases and two undescribed mannases. To better illustrate the relation of these 39 related proteins, four groups were created and analyzed by phylogenetic analysis: group A corresponding to xylanases, group B belonging to mannases and acting to degrade mannan; group C containing all known and putative cellulose-degrading proteins that have highly conserved CBMs; and group D containing beta-glucosidase and beta-xylosidase. Group D was the largest group, in which 8 beta-glucosidases appeared to be non-secreted proteins.     

Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.     

The use of canola meal as a substrate for xylanase production by Trichoderma reesei

Summary Tests made utilizing canola meal as a substrate for the production of xylanase indicate that Trichoderma reesei produced this enzyme in similar or better yields from canola meal than from Solka-floc, xylan or glucose. The maximum xylanase activity obtained from canola meal was 210 IU/ml in 9–12 days. The enzyme system produced using canola meal also contained a higher proportion of acetyl-xylan esterase, cellulase, and xylosidase activities. This system was more than or equally efficient as that produced using Solka-floc in hydrolysing canola meal, corn cobs, corn and wheat brans, straw, and larchwood xylan to fermentable sugars. Offprint requests to: Z. Duvnjak     

Cellulose-binding domain of endoglucanase III from Trichoderma reesei disrupting the structure of cellulose

The DNA fragment encoding the cellulose binding domain of endoglucanase III (CBD_{EG III}) from Trichoderma reesei was subcloned and expressed in E. coli. The CBD_{EG III} had a high affinity for cellulose. The morphological and structural changes of cellulose after treatment with CBD_{EG III} indicated a 17% decrease in number of hydrogen bonds and a 16.5% decrease in crystalline index. X-ray diffraction and IR spectra analyses indicated that the destabilization and breakage of the hydrogen bonds in crystalline cellulose accounted for the non-hydrolytic disruption of the structure of cellulose.     

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.     

Process optimization for the production of diosgenin with Trichoderma reesei

Based on the response surface methodology, an effective microbial system for diosgenin production from enzymatic pretreated Dioscorea zingiberensis tubers with Trichoderma reesei was studied. The fermentation medium was optimized with central composite design (3⁵) depended on Plackett–Burmann design which identified significant impacts of peptone, K₂HPO₄ and Tween 80 on diosgenin yield. The effects of different fermentation conditions on diosgenin production were also studied. Four parameters, i.e. incubation period, temperature, initial pH and substrate concentration were optimized using 4⁵ central composite design. The highest diosgenin yield of 90.57% was achieved with 2.67% (w/v) of peptone, 0.29% (w/v) of K₂HPO₄, 0.73% (w/v) of Tween 80 and 9.77% (w/v) of substrate, under the condition of pH 5.8, temperature 30 °C. The idealized incubation time was 6.5 days. After optimization, the product yield increased by 33.70% as compared to 67.74 ± 1.54% of diosgenin yield in not optimized condition. Scale-up fermentation was carried out in a 5.0 l bioreactor, maximum diosgenin yield of 90.17 ± 3.12% was obtained at an aeration of 0.80 vvm and an agitation rate of 300 rpm. The proposed microbial system is clean and effective for diosgenin production and thus more environmentally acceptable than the traditional acid hydrolysis.