Reesei Rut-C30 Research Articles

Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei

BackgroundFilamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production.ResultRecombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei.ConclusionBased on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.

Artificial zinc finger protein mediated cellulase production in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.

Role of the antioxidant defense system during the production of lignocellulolytic enzymes by fungi.

Fungi are used for the production of several compounds and the efficiency of biotechnological processes is directly related to the metabolic activity of these microorganisms. The reactions catalyzed by lignocellulolytic enzymes are oxidative and generate reactive oxygen species (ROS). Excess of ROS can cause serious damages to cells, including cell death. Thus, the objective of this work was to evaluate the lignocellulolytic enzymes produced by Pleurotus sajor-caju CCB020, Phanerochaete chrysosporium ATCC 28326, Trichoderma reesei RUT-C30, and Aspergillus niger IZ-9 grown in sugarcane bagasse and two yeast extract (YE) concentrations and characterize the antioxidant defense system of fungal cells by the activities of superoxide dismutase (SOD) and catalase (CAT). Pleurotus sajor-caju exhibited the highest activities of laccase and peroxidase in sugarcane bagasse with 2.6g of YE and an increased activity of manganese peroxidase in sugarcane bagasse with 1.3g of YE was observed. However, P. chrysosporium showed the highest activities of exoglucanase and endoglucanase in sugarcane bagasse with 1.3g of YE. Lipid peroxidation and variations in SOD and CAT activities were observed during the production of lignocellulolytic enzymes and depending on the YE concentrations. The antioxidant defense system was induced in response to the oxidative stress caused by imbalances between the production and the detoxification of ROS.

Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30.

The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ~70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, β-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol.

Engineering Trichoderma reesei Rut-C30 with the overexpression of egl1 at the ace1 locus to relieve repression on cellulase production and to adjust the ratio of cellulolytic enzymes for more efficient hydrolysis of lignocellulosic biomass

Cellulose hydrolysis is a synergetic process performed sequentially by different cellulolytic enzymes including endoglucanases, exoglucanases and glucosidases. Trichoderma reesei has been acknowledged as the best cellulase producer, but cellulase production by T. reesei through submerged fermentation is costly due to intensive energy consumption associated with the process for mixing and aeration, since non-Newtonian fluid properties are developed with mycelial growth. Therefore, engineering the ratio of cellulolytic enzymes in the cocktail for more efficient cellulose hydrolysis is an alternative strategy for reducing cellulase dosage and thus saving cost in enzyme consumption for cellulose hydrolysis. In this study, T. reesei QS305 with high endoglucanase activity was developed from T. reesei Rut-C30 by replacing the transcription repressor gene ace1 with the coding region of endoglucanase gene egl1. Compared to T. reesei Rut-C30, T. reesei QS305 showed 90.0% and 132.7% increase in the activities of total cellulases and endoglucanases under flask culture conditions. When cellulase production by T. reesei QS305 was performed in the 5-L fermentor, cellulases activity of 10.7 FPU/mL was achieved at 108 h, 75.4% higher than that produced by T. reesei Rut-C30. Moreover, cellulases produced by T. reesei QS305 were more efficient for hydrolyzing pretreated corn stover and Jerusalem artichoke stalk.

Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators

BackgroundCellulase can convert lignocellulosic feedstocks into fermentable sugars, which can be used for the industrial production of biofuels and chemicals. The high cost of cellulase production remains a challenge for lignocellulose breakdown. Trichoderma reesei RUT C30 serves as a well-known industrial workhorse for cellulase production. Therefore, the enhancement of cellulase production by T. reesei RUT C30 is of great importance.ResultsTwo sets of novel minimal transcriptional activators (DBDace2-VP16 and DBDcre1-VP16) were designed and expressed in T. reesei RUT C30. Expression of DBDace2-VP16 and DBDcre1-VP16 improved cellulase production under induction (avicel or lactose) and repression (glucose) conditions, respectively. The strain TMTA66 under avicel and TMTA139 under glucose with the highest cellulase activities outperformed other transformants and the parental strain under the corresponding conditions. For TMTA66 strains, the highest FPase activity was approximately 1.3-fold greater than that of the parental strain RUT C30 at 120 h of cultivation in a shake flask using avicel as the sole carbon source. The FPase activity (U/mg biomass) in TMTA139 strains was approximately 26.5-fold higher than that of the parental strain RUT C30 at 72 h of cultivation in a shake flask using glucose as the sole carbon source. Furthermore, the crude enzymes produced in the 7-L fermenter from TMTA66 and TMTA139 supplemented with commercial β-glucosidase hydrolyzed pretreated corn stover effectively.ConclusionsThese results show that replacing natural transcription factors with minimal transcriptional activators is a powerful strategy to enhance cellulase production in T. reesei. Our current study also offers an alternative genetic engineering strategy for the enhanced production of industrial products by other fungi.

Mn2+ modulates the expression of cellulase genes in Trichoderma reesei Rut-C30 via calcium signaling

BackgroundThe filamentous fungus Trichoderma reesei Rut-C30 is one of the most vital fungi for the production of cellulases, which can be used for biofuel production from lignocellulose. Nevertheless, the mechanism of transmission of external stimuli and signals in modulating cellulase production in T. reesei Rut-C30 remains unclear. Calcium is a known second messenger regulating cellulase gene expression in T. reesei.ResultsIn this study, we found that a biologically relevant extracellular Mn2+ concentration markedly stimulates cellulase production, total protein secretion, and the intracellular Mn2+ concentration of Rut-C30, a cellulase hyper-producing strain of T. reesei. Furthermore, we identified two Mn2+ transport proteins, designated as TPHO84-1 and TPHO84-2, indicating that they are upstream in the signaling pathway that leads to cellulase upregulation. We also found that Mn2+ induced a significant increase in cytosolic Ca2+ concentration, and that this increased cytosolic Ca2+ might be a key step in the Mn2+-mediated regulation of cellulase gene transcription and production. The utilization of LaCl3 to block plasma membrane Ca2+ channels, and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) to interrupt calcium signaling, showed that Mn2+ exerts the induction of cellulase genes via calcium channels and calcium signaling. To substantiate this, we identified a Ca2+/Mn2+ P-type ATPase, TPMR1, which could play a pivotal role in Ca2+/Mn2+ homeostasis and Mn2+ induction of cellulase genes in T. reesei Rut-C30.ConclusionsTaken together, our results revealed for the first time that Mn2+ stimulates cellulase production, and demonstrates that Mn2+ upregulates cellulase genes via calcium channels and calcium signaling. Our research also provides a direction to facilitate enhanced cellulase production by T. reesei.

Optimization of cellulolytic enzyme components through engineering Trichoderma reesei and on-site fermentation using the soluble inducer for cellulosic ethanol production from corn stover

BackgroundCellulolytic enzymes produced by Trichoderma reesei are widely studied for biomass bioconversion, and enzymatic components vary depending on different inducers. In our previous studies, a mixture of glucose and disaccharide (MGD) was developed and used to induce cellulase production. However, the enzymatic profile induced by MGD is still not defined, and further optimization of the enzyme cocktail is also required for efficient ethanol production from lignocellulosic biomass.ResultsIn this study, cellulolytic enzymes produced by T. reesei Rut C30 using MGD and alkali-pretreated corn stover (APCS) as inducer were compared. Cellular secretome in response to each inducer was analyzed, which revealed a similar enzyme profile. However, significant difference in the content of cellulases and xylanase was detected. Although MGD induction enhanced β-glucosidase production, its activity was still not sufficient for biomass hydrolysis. To overcome such a disadvantage, aabgl1 encoding β-glucosidase in Aspergillus aculeatus was heterologously expressed in T. reesei Rut C30 under the control of the pdc1 promoter. The recombinant T. reesei PB-3 strain showed an improved β-glucosidase activity of 310 CBU/mL in the fed-batch fermentation, 71-folds higher than that produced by the parent strain. Meanwhile, cellulase activity of 50 FPU/mL was detected. Subsequently, the crude enzyme was applied for hydrolyzing corn stover with a solid loading of 20% through separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation, respectively, for ethanol production. Better performance was observed in the SHF process, through which a total of 119.9 g/L glucose was released within 12 h for concomitant ethanol production of 54.2 g/L.ConclusionsThe similar profile of cellulolytic enzymes was detected under the induction of MGD and APCS, but higher amount of cellulases was present in the crude enzyme induced by MGD. However, β-glucosidase activity induced by MGD was not sufficient for hydrolyzing lignocellulosic biomass. High titers of cellulases and β-glucosidase were achieved simultaneously by heterologous expression of aabgl1 in T. reesei and fed-batch fermentation through feeding MGD. We demonstrated that on-site cellulase production by T. reesei PB-3 has a potential for efficient biomass saccharification and ethanol production from lignocellulosic biomass.

Anaerobic co‐digestion of rice straw and soybean straw to increase biogas production by pretreatment with trichoderma reesei RUT C30

This study aim to enhance biogas production for the co‐digestion of rice straw and soybean straw (RSS), pretreatment of fermentation materials by Trichoderma reesei RUT C30 were investigated. The pretreatments were done at 30°C for 72 h, and anaerobic digestion for 5 weeks. The results showed that the cumulative biogas yield from pretreatment co‐digestion group RSS (214.1 NmL/g TSsubstrate) was 318% higher than the untreated group CG RSS (51.2 NmL/g TSsubstrate). The cumulative methane yield from RSS was 94.3 NmL CH4/g TSsubstrate, which was 807% higher than that of untreated group (CG RSS 10.4 NmL CH4/g TSsubstrate), and 90% of potential biogas generation was reached in 17 days, which was 39% shorter than that of pretreatment mono‐digestion groups (28 days). These results suggested that rice and soybean straw co‐digestion exhibits a positive synergistic effect and Trichoderma reesei RUT C30 provides an effective method for improving biogas production. © 2017 American Institute of Chemical Engineers Environ Prog, 37: 1050–1057, 2018

Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose

BackgroundThe induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest.ResultsTrichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes’ coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8.ConclusionsRecombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral mutation. Moreover, the increased transcription levels of cellulase genes, the related transcription factors, and the MFS transporter CRT1 contribute to the outstanding cellulase production of SEU-7. Our research advances strain improvement to enhance the induction efficiency of soluble carbon sources to produce cost-effective cellulase and hemicellulase in industry.

Improvement of cellulase production in Trichoderma reesei Rut-C30 by overexpression of a novel regulatory gene Trvib-1

Trichoderma reesei is a widely used cellulase producer, and development of robust strains for improved cellulase production is of great interest. In this study, the gene Trvib-1 encoding a putative transcription factor was overexpressed in T. reesei Rut-C30, and effects on cellulase production by the manipulation as well as corn stover degradation by the crude enzyme were investigated. Cellulase production and protein secretion were significantly improved in the culture of the recombinant T. reesei Vib-1, which were 200% and 219%, respectively, higher than that produced by the parent strain. Cellulase induction was enhanced in the presence of pure cellulose as well as various soluble inducers. Glucose released from the pretreated corn stover hydrolyzed by the crude enzyme in the recombinant strain was improved 40%. These results indicate that the overexpression of Trvib-1 is a feasible strategy for producing cellulase to enhance bioconversion efficiency of lignocellulosic biomass.

Synergistic Fermentation of Camphor Leaves by Cellulolytic Fungus and Optimization of Reducing Sugar Production

The degradation of lignocellulosic materials requires the synergy of multiple enzymes. Synergistic fermentation would be an alternative to a complete enzyme system and consequentially enhance enzymatic production. The present study used cellulolytic fungus of Penicillium decumbens, Aspergillus niger C112, and Trichoderma reesei RutC-30 as the starting strains, to screen for the best combination and proportion of synergistic fermentation. The experiments of different combinations and inoculum ratios for synergistic fermentation were performed, and Box-Behnken designs were adopted to optimize the fermentation conditions. The best combination of strains was confirmed as T. reesei RutC-30 and P. decumbens with an 8:2 inoculum ratio. The maximum sugar concentration was 1.48 g/L with CMCase (17.53 U/mL) and FPase (4.78 U/mL) in this situation. Response surface methodology revealed the optimized parameters as a substrate concentration of 5.93% at 32.12 °C, and a fermentation time of 5.85 d with a predicted sugar concentration of 2.91 g/L. Under these conditions, a maximum sugar production of 2.96 g/L was achieved in verification experiments.

Soybean hull induced production of carbohydrases and protease among Aspergillus and their effectiveness in soy flour carbohydrate and protein separation

Soybean hull consists mainly of three major plant carbohydrates, i.e., cellulose, hemicellulose and pectin. It is inexpensive and a good potential substrate for carbohydrase production because it is capable of inducing a complete spectrum of activities to hydrolyze complex biomass. Aspergillus is known for carbohydrase production but no studies have evaluated and compared, among Aspergillus species and strains, the soybean hull induced production of various carbohydrases. In this study, A. aculeatus, A. cinnamomeus, A. foetidus, A. phoenicis and 11 A. niger strains were examined together with T. reesei Rut C30, another known carbohydrase producer. The carbohydrases evaluated included pectinase, polygalacturonase, xylanase, cellulase, α-galactosidase and sucrase. Growth morphology and pH profiles were also followed. Among Aspergillus strains, morphology was found to correlate with both carbohydrase production and pH decrease profile. Filamentous strains gave higher carbohydrase production while causing slower pH decrease. The enzyme broths produced were also tested for separation of soy flour carbohydrate and protein. Defatted soy flour contains about 53% protein and 32% carbohydrate. The enzymatic treatment can increase protein content and remove indigestible oligo-/poly-saccharides, and improve use of soy flour in feed and food. Protease production by different strains was therefore also compared for minimizing protein degradation. A. niger NRRL 322 and A. foetidus NRRL 341 were found to be the most potent strains that produced maximal carbohydrases and minimal protease under soybean hull induction.

Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved.

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production.

BackgroundThe conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear.ResultsThe T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed.ConclusionsThe T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of β-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0550-3) contains supplementary material, which is available to authorized users.

A biotech-systematic approach to select fungi for bioconversion of winery biomass wastes to nutrient-rich feed

Grape marc and lees are main waste biomass in wine industry, which contain substantial amount of carbohydrates and nutrients, but currently deliver considerable carbon emission through landfill in Australia. This study was aimed to develop a biotechnological systematic approach to select suitable fungi for bioconversion of the winery biomass wastes into animal feed. The biotech-systematic approach was developed through assessment of nutrient/carbon source accessibility of the fungi, understanding of their metabolic reactions in solid state fermentation, and nutritive value of the fungi-fermented grape marc. From 13 Aspergillus, Rhizopus, and Trichoderma fungal species, A. oryzae DAR 3699, A. oryzae RIB40 and T. reesei RUT C30 were determined as the best fungi due to their promising biochemical capabilities to enhance protein contents and in vitro digestibility of grape marc in mono- and co-cultivations. The mono-fungous-cultured SSF process used these selected fungi is able to convert grape marc to nutrient-rich feed by increasing the protein contents from ∼5% to 26% and the digestibility from ∼25% to over 50%. R. oligosporus 2710 with R. oryzae 6201 or T. viride 15719 showed promising protein enrichment to 18–23% and digestibility improvement by ∼50% in their co-cultivations. This preliminary bioengineering strategy will be useful for selecting microorganisms for the bioconversion of organic wastes to high valuable products. This “green cycle” bioprocess will be useful to promote the old-fashioned waste treatment technologies for the cleaner production in food processing industries.

Linking hydrolysis performance to Trichoderma reesei cellulolytic enzyme profile.

Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, for example, by spiking with single enzymes and monitoring hydrolysis performance. In this study, a multivariate approach, partial least squares regression, was used to see whether it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by T. reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed pretreated corn stover as a measure of enzyme performance. In addition, the enzyme mixtures were analyzed by liquid chromatography-tandem mass spectrometry to identify and quantify the different proteins. A multivariate model was applied for the prediction of enzyme performance based on the combination of different proteins present in an enzyme mixture. The multivariate model was used for identification of candidate proteins that are correlated to enzyme performance on pretreated corn stover. A very large variation in hydrolysis performance was observed and this was clearly caused by the difference in fermentation conditions. Besides β-glucosidase, the multivariate model identified several xylanases, Cip1 and Cip2, as relevant proteins to study further.

A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide

Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher β-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of β-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening.

BackgroundLignocellulase hypersecretion has been achieved in industrial fungal workhorses such as Trichoderma reesei, but the underlying mechanism associated with this process is not well understood. Although previous comparative genomic studies have revealed that the mutagenic T. reesei strain RUT-C30 harbors hundreds of mutations compared with its parental strain QM6a, how these mutations actually contribute to the hypersecretion phenotype remains to be elucidated.ResultsIn this study, we systematically screened gene knockout (KO) mutants in the cellulolytic fungus Neurospora crassa, which contains orthologs of potentially defective T. reesei RUT-C30 mutated genes. Of the 86 deletion mutants screened in N. crassa, 12 exhibited lignocellulase production more than 25% higher than in the wild-type (WT) strain and 4 showed nearly 25% lower secretion. We observed that the deletion of Ncap3m (NCU03998), which encodes the μ subunit of the adaptor protein 3 (AP-3) complex in N. crassa, led to the most significant increase in lignocellulase secretion under both Avicel and xylan culture conditions. Moreover, strains lacking the β subunit of the AP-3 complex, encoded by Ncap3b (NCU06569), had a similar phenotype to ΔNcap3m, suggesting that the AP-3 complex is involved in lignocellulase secretion in N. crassa. We also found that the transcriptional abundance of major lignocellulase genes in ΔNcap3m was maintained at a relatively higher level during the late stage of fermentation compared with the WT, which might add to the hypersecretion phenotype. Finally, we found that importation of the T. reesei ap3m ortholog Trap3m into ΔNcap3m can genetically restore secretion of lignocellulases to normal levels, which suggests that the effect of the AP-3 complex on lignocellulase secretion is conserved in cellulolytic ascomycetes.ConclusionsUsing the model cellulolytic fungus N. crassa, we explored potential hypersecretion-related mutations in T. reesei strain RUT-C30. Through systematic genetic screening of 86 corresponding orthologous KO mutants in N. crassa, we identified several genes, particularly those encoding the AP-3 complex that contribute to lignocellulase secretion. These findings will be useful for strain improvement in future lignocellulase and biomass-based chemical production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0302-3) contains supplementary material, which is available to authorized users.

Validation of a novel sequential cultivation method for the production of enzymatic cocktails from Trichoderma strains.

The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry.