Abstract:Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2.Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.

Abstract:Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2.Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.

Abstract:Abstract: [Objective] The aim of this study was to screen straw-cellulose degrading microorganisms and to investigate their degradation ability of straw-cellulose. [Methods] The methods used to screen the high effect straw-cellulose degrading microorganism included the traditional isolation methods of straw-cellulose degrading microorganism such as holes observation method on filter paper sheet, disintegration test of filter paper scrip, hydrolysis spot diameter measurement method of CMC-Na, weight lose assay method of straw, measurement method of cellulose decomposition rate, measurement method of extracellular enzyme activity. [Results] We isolated 3 fungi with cellulose degrading ability, of which 98MJ was identified as Penicillium oxalicum, W3 as Trichoderma sp., and W4 as Penicillium expansum. Strain W4 possessed high straw-cellulose degrading ability with straw-cellulose degrading rate of 56.3%, cellulose 59.06%,, hemicellulose 78.75% and lignin 33.79% in 10 days. [Conclusion] Strain W4 was a cellulase-producing strain with broad development potential.

Abstract:Abstract: Hypocrea jecorina（anamorph：Trichoderma reesei）is the main industrial fungi that can produce large amounts of extracellular cellulases and hemicellulases. It also represents a model system to study the mechanism of transcriptional regulation in eukaryotes. The expression of these hydrolases genes in Hypocrea jecorina can be triggered rapidly in the presence of inducers, but differences in the inducing mode of various soluble inducers have been reported. At present, three models have been offered to explain the question of “how an insoluble inducer such as cellulose would initiate the transcription of cellulases?” Moreover, interactions between the identified positive (Xyr1, Ace2, Hap2/3/5) and negative transcriptional regulators (Ace1, Cre1), as well as the interactions between these proteins and the promoters of cellelase and hemicellulase genes have also been primarily characterized. This review focuses on the key factors and the current understanding on the regulation of expression of cellulase and hemicellulase genes in Hypocrea jecorina.

Abstract:Abstract:Cellulosilyticum ruminicola H1 is an anaerobic cellulolytic bacterium isolated from the rumen content of yak．Previously，we found that strain H1 lost growth on filter paper cellulose after several subcultures．The growth on cellulose cannot be restored until a few transfers in the cellobiose-containing medium． This is contrary to the knowledge that microbial cellulases synthesis is induced by the substrate while repressed by the metabolites，e.g.cellobiose，but provides a hint of cell-density mediated cellulase synthesis．［Objective］ The study aimed to test if cell density regulation mechanism involved in the cellulase synthesis by Cellulosilyticum ruminicola H1．［Methods］By using enzyme assays and real-time PCR quantification，we investigated cellulase activities and the gene transcript levels in the high- and lowcellmasscultures of strain H1． We also determined the elevation of endoglucanase and cellobiohydrolase activities and the gene expression in the low-cellmass culture upon addition of the high-cellmass spent culture．［Results］ Both endoglucanase and cellobiohydrolase of strain H1 were detected 3 to 10 times higher in the high cellmass culture than in the lower cellmass culture either in enzymatic activity or the gene transcript abundance，thus confirming the cell densitymediated cellulase synthesis．［Conclusion］This study demonstrates that cell density regulation is involved in cellulases synthesis by Cellulosilyticum ruminicola H1．

Abstract:Objective To improve the overall quality of upper tobacco leaves.Methods We isolated a hemicellulase-producing bacterium from tobacco soil and identified it as Actinacidiphila bryophytorum. After optimizing the culture conditions of this strain, we prepared the crude enzyme liquid, which was sprayed on upper tobacco leaves. The physical properties and chemical composition of the treated tobacco leaves were then evaluated.Results The optimal fermentation conditions for the strain were an inoculation rate of 4%, pH 6.0, and incubation at 30 ℃ for 36 h. Enzyme preparations of different concentrations (50, 100, 150, 200 and 250 U/mL) were then sprayed on the surface of upper tobacco leaves, and the appearance quality, conventional chemical composition, lignocellulose content, and sensory scores of the tobacco leaves were evaluated. The results indicated that treatment of the enzyme preparation at 150 U/mL performed better than other treatments. Compared with the control group, the 150 U/mL treatment increased the overall score for the appearance quality (maturity, color, characteristics, and structure) of tobacco leaves by 5.15 points. Additionally, this treatment increased the total sugar content and reducing sugar content by 29.87% and 35.77%, respectively. Meanwhile, it decreased the content of nicotine, cellulose, hemicellulose, and lignin by 16.10%, 16.37%, 20.22%, and 17.13%, respectively. Furthermore, the aroma characteristics were enhanced, and off-flavors were reduced.Conclusion The enzyme preparation produced by the fermentation of A. bryophytorum S2 improves the appearance quality, increases the soluble total sugar and reducing sugar content, and reduces the lignocellulose content of tobacco leaves, thus improving the overall quality of tobacco leaves.

Abstract:Lignocellulose is a cheap, abundant and underutilized bioresource. Cellulases can break down cellulose into glucose that could further be fermented to bioethanol, thus has commercial potentials. The improvement of cellulase production helps the use of the abundantly available lignocellulose, benefits the further exploration of the potential of bioenergy, and assists the relief of the energy crisis. However, glycosylation has an important influence on catalytic activity, thermal stability and other properties. Therefore, the detailed knowledge on cellulase glycosylation is required. Reasonably maniputing cellulase glycosylation modifications can accelerate the hydrolysis of lignocellulose, and contribute to produce liquid biofuels.

Abstract:[Objective] In order to study carbon metabolic repressors on cellulase activities and expression regulation in Trichoderma reesei, we constructed multi-targeting siRNA expression vectors to perform silencing carbon metabolic repressor cre1, cre2, cre3 and cre4 by simultaneous producing multi-targeting siRNAs. [Methods] According to previous studies and screening, the four best siRNAs targeting the cre1, cre2, cre3 and cre4 were selected and they were constructed a multi-targeting siRNA expression vector A. Additionally, according to 5 overlap common sequence in the cre1, cre2, cre3 and cre4, we designed and constructed the vector B. The two vectors were transformed the protoplasm of Trichoderma reesei QM9414 and selected on the hygromycin selection medium. Cellulase activities (CMC activity and filter paper activity) of transformants were detected and related gene expressions were also detected by RT-qPCR after incubation for 48 and 120 hours respectively. [Results] RT-qPCR results showed that the cre1, cre2, cre3 and cre4 expression levels in Trichoderma reesei were simultaneously silenced, and the cellulase activities were much higher than that of the staring strain. The CMC activity and filter paper activity of the multi-targeted inhibitor strains were 1.95-fold and 2.66-fold higher than those of the original strains. The cellulase-related genes expression levels were also increased significantly. The expression levels of cbh1, egl1 and xyr1 were 3.83-fold, 3.95-fold and 2.78-fold higher than those of the original strain. [Conclusion] Our results indicated that simultaneous silencing multi-targets of the carbon metabolic repressors in Trichoderma reesei is beneficial to release the glucose repressor effects and increase the expression and production of cellulase. These results provide evidence and techniques for study of regulation of carbon catabolite repressor genes on cellulase expression in Trichoderma reesei.

Abstract:Abstract:［Objective］To isolate and identify the cellulose-producing bacterium from fresh feces of healthy giant pandas，and characterize its cellulase production． ［Methods］A strain with high activity of cellulase was isolated and purified by carboxymethyl cellulose-Na (CMC-Na) medium． The morphological，physiological and biochemical characteristics and 16S rDNA gene were analyzed to identify the taxonomic position of the strain． Meanwhile，its cellulase producing conditions and degradation of several cellulose substrates were studied． ［Results］A cellulose-producing strain P2 was obtained． Strain P2 is a gram-positive and aerobic bacterium． Its growth temperature ranges from 20 to 50℃ (optimum at 37℃) and pH 6.0 to 9.0 (optimum at 7.0)，and NaCl concentration at 0%－15% (optimum at 2.0%)． It took 24 h to reach the peak of cellulase production． Phylogenetic analysis based on 16S rDNA sequence showed that strain P2 is most closely related to the Bacillus amyloliquefaciens NBRC15535 with the similarity of 99. 66%． Strain P2 posses different abilities to degrade four different cellulose substrates including filter paper，absorbent cotton，straw，and bamboo fiber． During the degradation，it shows different enzymatic activity curves with endoglucanases，exoglucanases，β-glucosidases and cellulase． ［Conclusion］An aerobic cellulolytic bacterium was isolated from feces of giant pandas for the first time，and was identified as Bacillus amyloliquefaciens． Due to its ability to degrade the cellulose materials withdifferent structures，strain P2 could be used in the further study on the digestion mechanism of bamboo of giant panda．

Abstract:Conversion from lignocellulose to biofuel with enzyme hydrolysis will be beneficial for sustainable development. Most microorganisms used in the industrial production of cellulase are filamentous fungi. However, isolation of cellulase hyper producing strain is limited by the lack of proven technology on genetic modification of filamentous fungi and the mechanism of cellulase synthesis. In this paper, recent progress in the genetic modification of cellulase hyper producing strains are summarized. Specially, we discussed the influences of signal induction, signal transduction and transcriptional regulation on cellulase synthesis in filamentous fungi. Finally, the industrial application of filamentous fungi was also introduced.