Constitutive overexpression of cellobiohydrolase 2 in Trichoderma reesei reveals its ability to initiate cellulose degradation

Cellulose degradation results from the synergistic effect of different enzymes,but which enzyme is involved in the initial stage of cellulose degradation is still not well understood.Cellobiohydrolase 2(CBH2)attached to the conidial surface is possibly associated with the initial stage.However,its specific mechanism is still incompletely known.This study explored the potential role of CBH2 in initiating cellulose degradation using a constitutive overexpression strategy.First,the CBH2-overexpression Trichoderma reesei strains Qgc2-5 and Qrc2-40 were constructed using the constitutive promoters P gpd1 and P rpS30,respectively.It was found that cbh2 was ex-pressed at a high level under the glucose conditions and was significantly higher than that of the parental strain QM9414 at the early stage of 29 h when cellulose was used as the carbon source.Particularly,the constitutive overexpression of cbh2 caused the strong expression of major cellulase-encoding genes(cbh1,eg1,and eg2)and the rapid decomposition of cellulosic material.Meanwhile,the scanning electron microscope showed that the groove-like structure of the cellulose surface was eroded seriously owing to CBH2 overexpression,which caused the cellulose surface to be smooth.These results showed that the overexpression of CHB2 caused the major cel-lulase enzymes to be expressed and contributed to cellulose degradation,showing the potential role of CBH2 in the initial stage of the cellulose hydrolytic process.

Cumulative cellulolytic enzyme activities and initial litter quality in prediction of cellulose degradation in an alpine meadow of the eastern Tibetan Plateau

Aims Plant litter decomposition is a key ecosystem process that determines carbon and nutrient cycling in terrestrial ecosystems.As a main component of litter,cellulose is a vital energy source for the microbes associated with litter decomposition.The important role of cellulolytic enzymes in litter cellulose degradation is well understood,but seasonal patterns of cellulose degradation and whether cumulative enzyme activities and litter quality forecast cellulose degradation in an alpine meadow remain elusive,which limits our understanding of cellulose degradation in herbaceous plant litter.Methods A two-year field litterbag experiment involving three dominant species(Ajuga ovalifolia,Festuca wallichanica,and Pedicularis roylei)was conducted in an alpine meadow of the eastern Tibetan Plateau to explore the seasonal patterns of cellulose degradation and how cumulative cellulolytic enzyme activities and initial litter quality impact cellulose degradation.Important findings Our study demonstrates that cellulose degraded rapidly and exceeded 50%during the first year,which mainly occurred in the first growing season(31.9%–43.3%).At two years of decomposition,cellulose degradation was driven by cumulative endoglucanase(R^(2)=0.70),cumulative cellobiohydrolase(R^(2)=0.59)and cumulative 1,4-β-glucosidase(R^(2)=0.57).In addition,the concentrations of cellulose,dissolved organic carbon,total phenol,lignin and lignin/N accounted for 52%–78%of the variation in cellulose degradation during the two years of decomposition.The best model for predicting cellulose degradation was the initial cellulose concentration(R^(2)=0.78).The enzymatic efficiencies and the allocation of cellulolytic enzyme activities were different among species.The cellulolytic enzyme efficiencies were higher in the litter of F.wallichanica with relatively lower quality.For the complete cellulose degradation of the leaf litter,A.ovalifolia and F.wallichanica required 4-fold and 6.7-fold more endoglucanase activity,3-fold and 4.5-fold more cellobiohydrolase activity and 1.2-fold and 1.4-fold more 1,4-β-glucosidase activity,respectively,than those required by P.roylei.Our results demonstrated that although microbial activity and litter quality both have significant impacts on cellulose degradation in an alpine meadow,using cellulose concentration to predict cellulose degradation is a good way to simplify the model of cellulose degradation and C cycling during litter decomposition.

Screening and identification of cellulose-degradation strain M1 and its characteristics of cellulase

In this paper, in order to get the target microbe which has high cellulose bio-degradation ability, we collected soil samples from environment rich in cellulose. Carboxymethyl cellulose sodium gel plate and filter paper plate were used for microbe screening, and finally we got a strain named MI which has the highest cellulase producing ability, and it has been identified as Penicilium oxalicum by morphological and molecular biological identification. We have studied cellulose degradation ability of this strain under different conditions such as pH value, temperature and inoculating time. The results showed that CMCase which belongs to acid enzyme, could achieve its maximum enzyme activity 489.96 U/ml when the optimal pH value was 4. The FPAase could achieve its maximum enzyme activity 1,595.45 U/ml when the optimal pH value was 4, too. The optimal ferment temperature of CMCase is 50℃ when achieved the maximum of enzyme activity at the second day, whereas the FPAase could reach its tip top on the third day, So the optimal ferment temperature of FPAase is also 50℃.

Degradation of corn stalk by the composite microbial system of MC1

The composite microbial system of MC1 was used to degrade corn stalk in order to determine properties of the degraded products as well as bacterial composition of MC1. Results indicated that the pH of the fermentation broth was typical of lignocellulose degradation by MC1, decreasing in the early phase and increasing in later stages of the degradation. The microbial biomass peaked on the day 3 after degradation. The MC1 efficiently degraded the corn stalk by nearly 70% during which its cellulose content decreased by 71.2%, hemicellulose by 76.5% and lignin by 24.6%. The content of water-soluble carbohydrates （WSC） in the fermentation broth increased progressively during the first three days, and decreased thereafter, suggesting an accumulation of WSC in the early phase of the degradation process. Total levels of various volatile products peaked in the third day after degradation, and 7 types of volatile products were detected in the fermentation broth. These were ethanol, acetic acid, 1,2-ethanediol, propanoic acid, butanoic acid, 3- methyl-butanoic acid and glycerine. Six major compounds were quantitatively analysed and the contents of each compound were ethanol （0.584 g/L）, acetic acid （0.735 g/L）, 1,2-ethanediol （0.772 g/L）, propanoic acid （0.026 g/L）, butanoic acid （0.018 g/L） and glycerine （4.203 g/L）. Characterization of bacterial cells collected from the culture solution, based on 16S rDNA PCR-DGGE analysis of DNAs, showed that the composition of bacterial community in MC1 coincided basically with observations from previous studies. This indicated that the structure of MC1 is very stable during degradation of different lignocellulose materials.

Process of rice straw degradation and dynamic trend of pH by the microbial community MC1

The process of the rice straw degradation in the fermentor with aeration at 290 ml/h was studied. The results of dissolved oxygen （DO） indicated that the optimum DO during cellulose degradation by microbial community MC1 ranged from 0.01 to 0.12 mg/L. The change model ofpH values was as follows： irrespective of the initial pH of the medium, pH values decreased rapidly to approximate 6.0 after being inoculated within 48 h when cellulose was strongly degraded, and then increased slowly to 8.0--9.0 until cellulose was degraded completely. During the degradation process, 15 kinds of organic compounds were checked out by GC-MS. Most of them were organic acids. Quantity analysis was carried out, and the maximum content compound was ethyl acetate which reached 13.56 g/L on the day 4. The cellulose degradation quantity and ratio analyses showed that less quantity （under batch fermentation conditions） and longer interval （under semi-fermentation conditions） of rice straw added to fermentation system were contributed to matching the change model of pH, and increasing the quantity and ratio of rice straw degradation during cellulose degrading process. The highest degradation ratio was observed under the condition office straw added one time every five days （under semi-fermentation conditions）.

Screening of the effective cellulose-degradable strain and its application in the production of cellulose bioethanol

Strains from the cellulose-containing environment were collected. Primary screening(by filter-paper Hutchison solid culture medium and sodium carboxymethylcellulose solid culture medium) and reelection(by filter-paper inorganic salt culture medium and sodium carboxymethylcellulose Congo red culture medium) indicated that five strains obtained were best suited for high performance cellulose degradation. Determination of sodium carboxymethylcellulose activity(CMCA) and filter paper activity(FPA) was accomplished for each of the five. The strongest of the five in CMCA and FPA was applied to the production of cellulose bioethanol by separate hydrolysis and fermentation(SHF) and simultaneous saccharification and fermentation(SSF) respectively.

Soluble Carbohydrates Repress the Cellulolytic Activity of Clostridium cellulovorans and Eubacterium cellulosolvens

Studies have provided indirect evidence that cellulolytic activity of some anaerobic bacteria is repressed by carbohydrates, such as glucose. This effect is known as carbon catabolite repression （CCR）. Previous work has found that cellulolytic activity of Clostridium cellulovorans and Eubacterium cellulosolvens are regulated. Many cellulolytic systems of these organisms are expressed only in the presence of cellulose or cellobiose （the disaccharide of cellulose）. Some of these cellulose-induced systems also appear subject to CCR when more soluble substrates, such as glucose, are also available. To determine if such repression directly effects cellulolytic activity of C. cellulovorans and E. cellulosolvens, these organisms were cultivated in media containing a glucose analog. We then measured the ability of low levels of analog to inhibit growth of the organisms when cellobiose or cellulose were the energy substrates. Our results found that growth of both C. cellulovorans and E. cellulosolvens in cellobiose-containing medium are strongly inhibited by glucose analogs. In addition, both organisms exhibited delayed and slower growth in cellulose-containing medium when a glucose analog was added. These results provide direct demonstration that these cellulolytic bacteria are subject to CCR. This repression of cellulolysis may affect both of these organisms＇ ability to serve as industrial platforms for biomass degradation, and may interfere with the contribution of E. cellulosolvens toward animal digestion of cellulose. These results were also in sharp contrast to what has been reported regarding CCR activity in Clostridium cellulolyticum, which actively expresses cellulases in the presence of low levels of glucose.

A Peptide-mediated Fenton Reaction in Wood-degrading Fungi

Gt factor is a low-molecular-weight peptide isolated from the extracellular culture of wood-degrading fungus Gloeophyllum trabeum. It is capable of enhancing degradation of cellulose. Its action mechanism was investigated and it was found that Gt factor could reduce Fe3+ to Fe2+. Electron paramagnetic resonance (EPR) spectroscopy revealed in the presence of O2, Gt factor could drive the formation of H2O2 via a superoxide anion O2.- intermediate and mediate the generation of hydroxyl radical HO˙ in a Fenton-type reaction. All these provided evidence for the formation of HO˙ in some wood-degrading fungi.

Two-step fermentation process and the corresponding study on its enzymology characteristics

Strain HIT-3,which has good performances in cellulose degradation,was isolated from the campus soil. Based on the identification of conservative sequences 16S rDNA and the analysis of physiological-biochemical characteristics,HIT-3 was identified as Achromobacter xylosoxidans. Denitrificans. A two-step fermentation process was conducted by adopting compound-bioflocculant-producing flora constructed by cellulose-degrading bacterium HIT-3 and flocculating bacterium F2. The cellulose degradation metabolites of HIT-3 was taken as substrates by flocculating bacterium F2,by which excellent compound bio-flocculant was obtained. In addition,the enzymology characteristics of HIT-3 were investigated when cultured in cellulose media,which utilized CMC-Na as its sole carbon. The results show that HIT-3 achieves a climax of 67.6 U/mL of enzyme production after incubation for 6 d,and the organic carbons produced are sufficient as the substrates required by the fermentation of flocculating bacterium F2(flocculating efficiency of 85.6%),which makes it feasible to reuse bioenergy.

Phylogenetic Analysis of Anaerobic Co-Digestion of Animal Manure and Corn Stover Reveals Linkages between Bacterial Communities and Digestion Performance

Over 3 million tons of manures are produced annually in the United States and pose environmental and health risks if not remediated. Anaerobic digestion is an effective method in treating organic wastes to reduce environmental impacts and produce methane as an alternative energy. Previous studies suggested that optimization of feed composition, hydraulic retention time, and other operational conditions can greatly improve total solids removal and increase methane productivity. These environmental factors improve functionality by altering the microbial community structure but explicit details of how the bacterial community shifts are poorly understood. Our investigations were conducted to investigate the relationship between environmental factors, microbial community structure and bioreactor efficiency by using metagenomic analysis of the microbial communities. Our results indicated that the bioreactor with the greatest methane production, digestion efficiency and reduced levels of E. coli/Shigella had a distinctive community structure at the genus level with unique and abundant uncultivated strains of Bacteroidetes. Moreover the same bioreactor was enriched in Aminomonas paucivorans and Clostridia populations that can utilize secondary metabolites produced during cellulose/hemicellulose degradation to generate hydrogen and acetate. Hence specific digestion conditions that enrich for these populations may provide a route to the optimization of co-digestion systems and control the variability in reactor performance.

Characterization of the effective cellulose degrading strain CTL-6

An efficient cellulose degrading bacteria exists in the thermophilic wheat straw-degrading community, WDC2. However, this strain cannot be isolated and cultured using conventional separation techniques under strict anaerobic conditions. We successfully isolated a strain of effective cellulose degrading bacteria CTL-6 using a wash, heat shock, and solid-liquid alternating process. Analysis of its properties revealed that, although the community containing the strain CTL-6 grew under aerobic conditions, the purified strain CTL-6 only grew under anaerobic culture conditions. The strain CTL-6 had a striking capability of degrading cellulose （80.9% weight loss after 9 days of culture）. The highest efficiency value of the endocellulase （CMCase activity） was 0.404 μmol/（min·mL）, cellulose degradation efficiency by CTL-6 was remarkably high at 50–65°C with the highest degradation efficiency observed at 60°C. The 16S rRNA gene sequence analysis indicated that the closest relative to strain CTL-6 belonged to the genus Clostridium thermocellum. Strain CTL-6 was capable of utilizing cellulose, cellobiose, and glucose. Strain CTL-6 also grew with Sorbitol as the sole carbon source, whereas C. thermocellum is unable to do so.

Affinity-induced covalent protein-protein ligation via the SpyCatcherSpyTag interaction

Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.

The type IX secretion system: Insights into its function and connection to glycosylation in Cytophaga hutchinsonii

The recently discovered type IX secretion system(T9SS)is limited to the Bacteroidetes phylum.Cytophaga hutchin-sonii,a member of the Bacteroidetes phylum widely spread in soil,has complete orthologs of T9SS components and many T9SS substrates.C.hutchinsonii can efficiently degrade crystalline cellulose using a novel strategy,in which bacterial cells must be in direct contact with cellulose.It can rapidly glide over surfaces via unclear mech-anisms.Studies have shown that T9SS plays an important role in cellulose degradation,gliding motility,and ion assimilation in C.hutchinsonii.As reported recently,T9SS substrates are N-or O-glycosylated at their C-terminal domains(CTDs),with N-glycosylation being related to the translocation and outer membrane anchoring of these proteins.These findings have deepened our understanding of T9SS in C.hutchinsonii.In this review,we focused on the research progress on diverse substrates and functions of T9SS in C.hutchinsonii and the glycosylation of its substrates.A model of T9SS functions and the glycosylation of its substrates was proposed.