Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.     

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.     

Secretome analysis of the fungus Trichoderma harzianum grown on cellulose

Trichoderma harzianum is a mycoparasitic filamentous fungus that produces and secretes a wide range of extracellular hydrolytic enzymes used in cell wall degradation. Due to its potential in biomass conversion, T. harzianum draws great attention from biofuel and biocontrol industries and research. Here, we report an extensive secretome analysis of T. harzianum. The fungus was grown on cellulose medium, and its secretome was analyzed by a combination of enzymology, 2DE, MALDI-MS and -MS/MS (Autoflex II), and LC-MS/MS (LTQ-Orbitrap XL). A total of 56 proteins were identified using high-resolution MS. Interestingly, although cellulases were found, the major hydrolytic enzymes secreted in the cellulose medium were chitinases and endochitinases, which may reflect the biocontrol feature of T. harzianum. The glycoside hydrolase family, including chitinases (EC 3.2.1.14), endo-N-acetylglucosaminidases (EC 3.2.1.96), hexosaminidases (EC 3.2.1.52), galactosidases (EC 3.2.1.23), xylanases (EC 3.2.1.8), exo-1,3-glucanases (EC 3.2.1.58), endoglucanases (EC 3.2.1.4), xylosidases (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), N-acetylhexosaminidases (EC 3.2.1.52), and other enzymes represented 51.36% of the total secretome. Few representatives were classified in the protease family (8.90%). Others (17.60%) are mostly intracellular proteins. A considerable part of the secretome was composed of hypothetical proteins (22.14%), probably because of the absence of an annotated T. harzianum genome. The T. harzianum secretome composition highlights the importance of this fungus as a rich source of hydrolytic enzymes for bioconversion and biocontrol applications.     

Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses

PfMig1⁸⁸, a catabolically derepressed engineered strain of the hyper-cellulolytic fungus Penicillium funiculosum NCIM1228, was investigated for the efficacy of its secretome for biomass saccharification. An inexpensive version of media containing microcrystalline cellulose, wheat bran and soya protein was optimized for producing a high-quality secretome from the PfMig1⁸⁸ strain in both shake flasks and in a 20-L bioreactor. The activities of four classes of core cellulolytic enzymes required for saccharification in the PfMig1⁸⁸ secretome, namely, cellobiohydrolase (Avicelase activity), endoglucanase (CMCase activity), β-glucosidase (PNPGase activity) and xylanase (xylanase activity), were found to be 2.29 U/mL, 28.24 U/mL, 150 U/mL and 76 U/mL, respectively. The saccharification potential of the PfMig1⁸⁸ secretome was evaluated on rice straw and sugarcane bagasse pretreated with nitric acid and/or ammonium hydroxide. Saccharification performed using a 15 % (w/v) biomass load and a 3% (w/w) enzyme load released >100 g/L sugar in the hydrolysate, irrespective of the type of biomass and pre-treatment, with >80 % hydrolysis. Furthermore, the presence of lignin in nitric acid-pretreated biomass only marginally affected saccharification. Overall, the results demonstrated that the PfMig1⁸⁸ secretome, having relatively broad substrate specificity, is a viable and efficient substitute for T. reesei-based secretomes for diverse biomass saccharification.     

Comparative secretome analysis of <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> S4F8 and <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C30 during solid-state fermentation on sugarcane bagasse

Background

The lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).

Results

Comparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome.

Conclusions

In SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.

     

Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs

Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Autoclaved mycelium induces efficiently the production of hydrolytic enzymes for protoplast preparation of autologous fungus

The production of hydrolytic enzymes by the mutant Trichoderma reesei Rut-C30 when cultivated in the presence of various carbon sources: glucose, wheat bran and autoclaved mycelium of Penicillium occitanis CT1 has been studied. Glucose was shown to repress all studied hydrolases, 3% of either wheat bran or autoclaved cell walls led to high titers of enzymes, and were favorably comparable to commercial lysing enzymes (LE). The lysing enzyme cocktail obtained when T. reesei Rut-C30 was cultivated in the presence of autoclaved P. occitanis CT1 mycelia appeared to be a most effective for P. occitanis CT1 protoplast formation. Maximal yield of protoplasts reached 13 × 10⁶ protoplasts/mL while commercial LE preparation released only 4 × 10⁶ protoplasts/mL. The protoplast yield was affected also by the osmotic stabilizer, with KCl giving the best results. Our results suggest that to achieve the best protoplastization rate, the enzyme preparation should be obtained following induction by the autoclaved mycelium of the autologous fungus.     

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant‐degrading enzymes can be produced by fungi on abundantly available low‐cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi‐)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non‐washed SCB. The sensitivity to non‐washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.     

Biotransformation of Trichoderma spp. and Their Tolerance to Aromatic Amines,a Major Class of Pollutants

Trichoderma spp. are cosmopolitan soil fungi that are highly resistant to many toxic compounds. Here, we show that Trichoderma virens and T. reesei are tolerant to aromatic amines (AA), a major class of pollutants including the highly toxic pesticide residue 3,4-dichloroaniline (3,4-DCA). In a previous study, we provided proof-of-concept remediation experiments in which another soil fungus, Podospora anserina, detoxifies 3,4-DCA through its arylamine N-acetyltransferase (NAT), a xenobiotic-metabolizing enzyme that enables acetyl coenzyme A-dependent detoxification of AA. To assess whether the N-acetylation pathway enables AA tolerance in Trichoderma spp., we cloned and characterized NATs from T. virens and T. reesei. We characterized recombinant enzymes by determining their catalytic efficiencies toward several toxic AA. Through a complementary approach, we also demonstrate that both Trichoderma species efficiently metabolize 3,4-DCA. Finally, we provide evidence that NAT-independent transformation is solely (in T. virens) or mainly (in T. reesei) responsible for the observed removal of 3,4-DCA. We conclude that T. virens and, to a lesser extent, T. reesei likely utilize another, unidentified, metabolic pathway for the detoxification of AA aside from acetylation. This is the first molecular and functional characterization of AA biotransformation in Trichoderma spp. Given the potential of Trichoderma for cleanup of contaminated soils, these results reveal new possibilities in the fungal remediation of AA-contaminated soil.     

Secretome analysis identifies potential virulence factors of Diplodia corticola,a fungal pathogen involved in cork oak (Quercus suber) decline

The characterisation of the secretome of phytopathogenic fungi may contribute to elucidate the molecular mechanisms of pathogenesis. This is particularly relevant for Diplodia corticola, a fungal plant pathogen belonging to the family Botryosphaeriaceae, whose genome remains unsequenced. This phytopathogenic fungus is recognised as one of the most important pathogens of cork oak, being related to the decline of cork oak forests in the Iberian Peninsula.Unfortunately, secretome analysis of filamentous fungi is limited by the low protein concentration and by the presence of many interfering substances, such as polysaccharides, which affect the separation and analysis by 1D and 2D gel electrophoresis. We compared six protein extraction protocols concerning their suitability for further application with proteomic workflows. The protocols involving protein precipitation were the most efficient, with emphasis on TCA–acetone protocol, allowing us to identify the most abundant proteins on the secretome of this plant pathogen. Approximately 60 % of the spots detected were identified, all corresponding to extracellular proteins. Most proteins identified were carbohydrate degrading enzymes and proteases that may be related to D. corticola pathogenicity.Although the secretome was assessed in a noninfection environment, potential virulence factors such as the putative glucan-β-glucosidase, neuraminidase, and the putative ferulic acid esterase were identified.The data obtained forms a useful basis for a deeper understanding of the pathogenicity and infection biology of D. corticola. Moreover, it will contribute to the development of proteomics studies on other members of the Botryosphaeriaceae.     

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS‐MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono‐oxygenases (AA9), carbohydrate‐binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two‐fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327–336, 2016     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

Profiling of differentially expressed genes in ectomycorrhizal fungus Pisolithus tinctorius responding to mycorrhiza helper Brevibacillus reuszeri MPt17

A cDNA library of the ectomycorrhizal (ECM) fungus Pisolithus tinctorius Pt2 after interaction with the mycorrhiza helper bacterium (MHB) Brevibacillus reuszeri MPt17 was constructed by suppression subtraction hybridization. Total RNA from B. reuszeri MPt17 exudates treated Pi. tinctorius Pt2 was used as a “tester” and total RNA from nonbacterial treated Pi. tinctorius Pt2 was used as a “driver.” Among the differentially expressed sequences, a BLASTX in the NCBI non-redundant protein sequence database revealed that 75% of the non-redundant sequences (147 out of 196) were highly similar to known proteins (E-value < e^?5). Twelve sequences were annotated as mycelium development function combining with a potential functional categories using gene ontology. Quantitative real-time PCR analysis showed that all of the 3 symbiosis regulated acidic polypeptide genes were all up-regulated in the MPt17-treated Pt2. These results provide evidence that the MHB B. reuszeri MPt17 could significantly change the expression of symbiosis-related genes and genes in mycelium development in ECM fungus, and also support the hypothesis that the MHB functions as helper though promotion on fungal mycelium.     

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.     

Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.     

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.