Functional Analysis Of Cellulose Degradation Factor Swollenin And β-glucosidase In Trichoderma Reesei

As the most abundant biomass in nature, its decomposition not only plays a key role in the carbon cycle of nature, but also provides a great potential for a number of applications, most notably biofuel production. However, the bioconversion of cellulosic biomass into biofuels has remained a challenge largely due to the high cost of the whole process. Among others, the large quantities of cellulases required in the eff icient hydrolysis of lignocellulose represent a primary hindrance.Trichoderma reesei possesses one of the best-known cellulase enzyme systems, and it has served as a model for fungal cellulose degradation. Three categories of cellulases, endoglucanases, cellobiohydrolases andβ-glucosidases, are produced by saprophytic filamentous fungi for the degradation of insoluble cellulose into glucose. The endoglucanases mainly hydrolyze internal bonds in the cellulose polymer, producing new chain ends. The cellobiohydrolases, also known as exoglucanases, act processively from the chain ends, mainly producing cellobiose. This disaccharide and other short cello-oligomers are broken down to glucose byβ-glucosidases. The cellobiohydrolases and the endoglucanases from this fungus have been especially extensively characterized. Theβ-glucosidases of T. reesei, however, are less well characterized. Since genome sequence of T. reesei were open in 2008, sevenβ-glucosidases have been identified. The transcription level assays showed expression ofβ-glucosidases was different between higher and lower cellulase-producing strains of T. reesei. Although it is known thatβ-glucosidase activity is required for complete digestion of carbohydrates to monosaccharides, the precise functions and specificities of the enzymes encoded by these genes is not known.A variety of enzyme and non-enzyme components participate in cellulose degradation. Although the synergistic effects of cellulolytic enzymes such as cellobiohydrolases and endoglucanases have been relatively well studied, no clear relationships between cellulololytic and non-hydrolytic proteins with uncertain roles have emerged. However, some non-hydrolytic proteins such as plant expansins and expansin-like proteins produced by fungi and nematodes are known to be capable of loosening or disrupting the packaging of the plant cell wall and polysaccharides (such as cellulose and hemicellulose). Swollenin from fungi has been shown to be able to swell the cotton fibers and filter paper without producing detectable amounts of reducing sugars. Because of its unique characteristics, great interests have thus been provoked regarding its potential synergism with cellulases in the efficient hydrolysis of cellulose. Nevertheless, few researches on swollenin and its possible synergism with cellulases are taken in efficient hydrolysis of cellulose. Therefore, more information about the exact effect of swollenin and its possible synergism with cellulases in lignocellulose hydrolysis is required.Based on these two aspects, we constructed the expression system whose host was Aspergillus niger. the recombinant swollenin (SWO2) from T. pseudokoningii S38 was successfully heterologously expressed and purified in A. niger. The recombinant SW02 was further characterized as for its hydrolytic activities, and its potential synergistic action with cellulases was also evaluated. The transformants of truncted domain protein was obtained, whlie the mutant protein was not purified because of varied complicated factors. Cell a and cel1b knockout strains were constructed by homologous gene targeting. Function analysis of cel1a and cel1b was assessed inΔcel1a and Acel1b strains compared to the wild-type parental strain QM9414. Cel1b was heterologously expressed in E.coli, and characterized as for its hydrolysis activities and transglycosylation. The main results are as follows:1. The versatile expression vector was constructed with multiple cloning sites. The whole swollenin and truncted domain protein were expressed in A. niger.To achieve the high-level expression of the target protein, the expression plasmid was constructed under the control of glucamylase gene (glaA) promoter from A. niger, and HisHA tag was fused at the C-terminus of the promoter and followed by multiple cloning sites which were inserted by the whole swollenin and truncted domain protein. The pyrG cassette used as a selective marker was further ligated into expression plasmid. The expression plasmids were transformed into A. niger MGG029 mediated by PEG. Transformants were selected on uridine-minus plates and purified after a series of single-spore isolations. Purified transformants were further screened for the SW02 expression in maltose minimal medium by western blotting of the cultural supernatant. Transformants with relatively higher expression level were picked out and amplified for large-scale purification.2. Swollenin was purified to homogeneity from the culture supernatant of A. niger MGG029. Synergism for SWO2 and cellulases was analized in cellulose hydrolysis.The Swollenin was purified to near homogeneity from this concentrated supernatant by a two-step purification procedure involving anion exchange chromatography and hydrophobic chromatography with a final yield of about 10 mg Swollenin purified from 1 L of fermentation supernatant. The fraction eluted on every step was analized by SDS-PAGE and western blotting. The identity of the purified protein was confirmed by MS/MS sequencing. The observed molecular weight only slightly decreased as indicated by its faster motility on SDS-PAGE. This suggests that the difference between the calculated and observed molecular mass can not be simply explained by N-glycosylation, and that O-glycosilation and/or other unidentified modifications may contribute to this difference. Although hardly any hydrolysis could be observed in zymogram assay, the purified swollenin displayed a low RACase activity (0.107 U/mg) and an even lower CMCase activity (0.067 U/mg). Although low CMCase (2.6 U/mg) was also reported in the purified AfSwo1, we can not at present exclude the possibility of trace contamination of endoglucanase activities in the swollenin during the purification. To evaluate the potential effect of SWO2 with cellulase mixtures in promoting cellulose saccharification, crystalline cellulose, Avicel, was treated with SWO2 and cellulases either simultaneously or sequentially. The highest synergistic activity of sequential treatment as observed with swollenin is twice of that when cellulose was treated simultaneously with the same concentrations of swollenin and cellulases. Sequential treatment would otherwise make the somehow modified surface of cellulose resulting from swollenin treatment more readily hydrolyzable by the followed cellulases. 3. Cellb was heterologous expressed in Escherichia coli and potentially important residues at the catalytic site were changed by site-directed mutagenesis in order to dissect the catalytic mechanism of hydrolysis and transglycosylation.Based on the hydrolytic activity, Cellb appeared to be aβ-glucosidase. Also, Cel1b can hydrolyze sorphorose and the product was glucose, indicating that it can hydrolyzeβ-1,2 glycosidic bond. Several of family 1β-glucosidases have been shown to possess transglycosylation, and research into their use in oligosaccharide synthesis is being carried out. Cel1b also showed clear transglycosylation activity in our experiments, which were performed with high concentrations of cellobiose and glucose. The trials carried out in this work remained some optimization. Although Cel1a can produce sorphorose in previous report, sorphorose was not detected in the product of Cellb. For one thing, sorphorose and cellobiose overlapped on the Bio-Rad Aminex HPX-42A carbohydrate column so that both of them were not distinguished; for another, sorphorose was not produced. The structure of Cellb was obtained by homology moldling based on the crystle structure of Cel1a. Alignment of Cellb and others in family 1β-glucosidases showed residues around the glycone are highly conserved but those around the aglycone binding site were not conserved. Based on above analysis, five site-directed mutagenesis were substituted in new protein. As is shown in results, mut1 (I174C) surprisingly showed dramatic increase in hydrolytic activity, whose specific activity was 143 times than the wild type. Cys174 maybe play the important role in Cellb catalysis according to the structural analysis and kinetic parameter. Also, the exciting change of hydrolytic activity indicated a significant way to construct strains of high-producing cellulases and high efficiency cellulse system.4. Cella and cellb were deleted in order to clarified the role of them in cellulase induction.TheΔcel1a::pyr4 andΔcel1b::pyr4 targeting cassette were constructed and transformed into T. reesei by homologous integration. Transformants were shown to be deleted in the cel1a and cellb gene. The growth behavior of control strain QM9414 and the deletion strains of cel1a and cel1b was tested on cellobiose plates and Avicel plates. Compared to QM9414, theΔcel1a andΔcel1b growed slowly. Also, hydrolysis circle ofΔcel1a andΔcel1b was weaker than the control strain QM9414, especially cel1a. One possible explanation of these observations is that, by deleting cel1a or cel1b reducing intracellular metabolism of cellobiose, metabolism of cellobiose was repressed. The effect of disruption of cel1a and cel1b on protein export and cellulase production was determined in MA medium with Avicel as a carbon. Total protein production and exoglucanase activity were measured from QM9414,Δcel1a andΔcel1b. The gene deletion strains produced less protein than control strain at the early time during growth. Also, so is exoglucanase activity. It was different that the activity of three strains reached a top peak from protein expression. Exoglucanase production of control strains was significantly higher thanΔcel1a andΔcel1b.during both inducers. The mRNA levels of the CBHI showed a corresponding lag in induction, suggesting that the absence of cel1a and cel1b has an effect on the co-ordinate regulation of the other cellulose genes at the level of transcription. Accorsding to above analysis, deletion of cel1a and cel1b can delay induction of cellulase.