| Filamentous fungi Trichoderma reesei is famous for its high capacity to secret large amounts of glycoside hydrolase enzymes that act synergistically to release fermentable sugars from cellulose. Although its genome sequencing has been completed, there is still not enough information to fully illustrate the mechanism of its highly secretion ability. Cellobiohydrolase I (CBH1) represents the highest secreted cellulase component, typically making up to 60% of the total secreted proteins. Many cellulases produced by T. reesei, including CBH1, are multimodular with carbohydrate-binding modules (CBD) and catalytic domains (CD) connected by flexible, O-glycosylated linkers.N-linked glycosylation modulates and diversifies the structures and functions of the eukaryotic proteome through both intrinsic and extrinsic effects on proteins, such as altering structure and activity or enhancing proper folding and reduce protein aggregation or maintaining protein stability. N-glycosylation occurs at the correct site is a prerequisite of the glycoprotein correctly folding. N-glycosylation of CBH1 occurs at three (Asn45, Asn270 and Asn384) of the four potential N-glycosylation sites in catalytic domain. While Asn45 is located at the entrance of the catalytic tunnel, Asn270 is located near the distal end of the catalytic tunnel corresponding to the exit for the released cellobiose. Asn384, on the other hand, is located on a peptide loop at the bottom of the enzyme, which partially forms the catalytic tunnel on the loop region and thus affects the release of cellobiose from catalytic tunnel. It is still unknown the importance of the N-glycan on these sites for CBH1 secretion, secondary structure, enzyme activity and stability.The effects of N-glycosylation on heterologously expressed T. reesei CBH1 have been studied to some extent, and there is not homologously study yet. Because the type and extent of CBH1 N-glycan varied from host strains and culture conditions, and thus may disturb its hydrolytic activity and protein stability. In order to investigate the role of N-glycosylation for CBH1 folding, secretion, stability and enzyme activity in T. reesei, we constructed a series of N-glycosylation mutant variants by individual or combinatorial targeted change of asparagine at sites 45,270 and 384 to glutamine. Further analysis of CBH1 secreted into the culture supernatant from these mutant and wild type strains were conducted. Meanwhile, WT and mutant CBH1s were purified respectively to analyze whether their exoglucanase activity and protein stability were affected. The main results are as follows:1. Construction of Acbhl, a series of CBH1 N-glycosylation mutant strains and disruption of cnel gene in T. reesei WT and MT.In order to construct CBH1 N-glycosylation mutant strains, the cbhl gene was knocking out by homologous recombination with pyrG as the selective marker. The expression plasmid was constructed under the control of the endogenous cbhl promoter and the trpC terminator from A. niger. All the cbhl fragments with targeted mutations to eliminate N-glycosylation by replacing asparagine with glutamine were generated by site-directed mutagenesis using overlap-extension PCR. The expression plasmids were transformed into Acbhl, the pyrG gene introduced into Acbhl strain was replaced by the coding sequence of cbhl with mutations to eliminate N-glycosylation at respective site as indicated, transformants were selected on hygromycin (120μg/mL) and 5-FOA (1.2 mg/mL) plates, and purified after a series of single-spore isolations. Purified transformants were further screened and verified by PCR, DNA sequencing and Southern blot. Thus lay a solid foundation for further study the relationship between N-glycosylation and CBH1 secretion, protein stability or enzyme activity. The cnel disruption vector was also obtained via Bp-cloning and introduced to T. reesei WT and MT strains, respectively.2. The absence of N-glycosylation of CBHl has little effect on its extracellular secretion but induces expression of the UPR target genes.Elimination of N-linked glycosylation at either single or double even triple sites had hardly any effect on the extracellular level of CBH1, but the expression of UPR target genes was modulated to adapt to the folding of unglycosylated CBH1. The transcript level of ER chaperones (cnel) and UPR target gene (pdil and bipl) were increased, but the transcript level of these gene were not modulated in M45 M270 and M384 variants. Moreover, deletion of cnel had hardly any effect on the secretion of WT CBH1, but its absence drastically compromised the secretion of MT CBH1. Furthermore, we investigated the mobility of different CBH1 variants on SDS-PAGE and suspected that N-glycosylation form at N45 and N384 sites was GlcNAc while N270 was (ManP)0-1GlcMan7-8GlcNAc2 or (Man1-2P)0-1-2Man5-6-7GlcNAc2, respectively.3. Purified the WT and mutant CBHl proteins from fermentation liquor and found that the absence of N-glycosylation in CBHl does not significantly decrease its catalytic activity but N-glycosylation at N384 and N45 is important for the thermal reactivity of CBHl.To study the N-glycosylation mutant CBH1s, protein purification protocol was explored. The fermentation liquor of T. reesei was purified by ammonium sulfate precipitation, desalting by G25, anion exchange chromatography DEAF FF and Mono Q successively. The fractions eluted on each step were analyzed by SDS-PAGE and western blot. The identity of the purified protein was confirmed by MS/MS sequencing. Next, purified WT and mutant CBH1 proteins were used to analyze the relationship between N-glycosylation on different sites and protein stability and activity. We found that the absence of N-linked glycosylation of purified CBH1 does not significantly influence its catalytic activity with both soluble (pNPC) and insoluble (Avicel) substrates. But three N-glycosylation sites played different role in maintaining CBH1 thermal stability. As compared with N270, removal of either N45 which is located at the entrance of the catalytic tunnel or N384 which is located on a peptide loop at the bottom of the enzyme yields a mutant with a thermal labile property in catalyzing soluble substrate. Combined loss of these two N-linked glycans exacerbates further the temperature-dependent inactivation. Further CD spectra analysis revealed that, while removal of N384 site is more prone to result in a drastic change of the secondary structure upon temperature elevation, mutation at N45 had a less pronounced effect on the overall regular secondary structure. Therefore, as compared with glycosylation at N45, the thermal stabilizing effect conferred by glycosylation at Asn384 seems to result from its contribution to maintaining the secondary structure of the enzyme. The observed thermal instability of glycosylation deficient mutants is much less significant when catalyzing insoluble cellulose, suggesting that conformation of the bound unglycolsylated enzyme is more resistant to thermal inactivation. Moreover, unlike temperature, elimination of N-linked glycans did not lead to any alterations in the secondary structure under high pH condition though exoglucanase activity of CBH1 was nearly abolished. These data demonstrate a differential and temperature-specific influence of N-linked glycans on the structural stability of CBH1.4. a-mannosidase (mnslp) and ER degradation enhancing a-mannosidase like protein (EDEM1, mnllp) were deleted in order to clarified the role of them in cellulase induction.The Amns1p::pyrG and Amnllp::hph targeting cassette were constructed and transformed T. reesei TU6 by homologous integration. Transformants were verified by anchor PCR and Southern blot, and mnslp and mnllp gene were knockout successfully. There were no differences of the microscopic images and also growth behavior on the plates with different carbon sources between control strain TU6 and the recombinant strains Amnslp and Amnllp. Meanwhile, knockout of either mnslp or mnl1p gene hardly affected the extracellular level of CBH1. The hydrolytic activity of Amnslp extracellular supernatant toward Avicel was improved while Amnllp reduced, but both of their specific activities were lower than that of wild-type. Moreover, the specific activity of Amnslp extracellular supernatant was higher than that of TU6 on pNPC, but there were no difference in CMC hydrolytic activity between wild type and recombinants. Then CBH1 proteins were purified from recombinant strains Amnslp and Amnllp, and two N-glycosylation forms of CBH1 were found. Further study revealed that there was no difference of the hydrolysis activity towards pNPC and Avicel for CBH1s with different N-glycosylation forms. But their thermal transition temperatures were different, the CBH1 with high-mannose oligosaccharide N-glycans was more stable than that of with simple N-glycans, which shows that the length and composititon of N-glycan might affect the thermostability of CBH1. |
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