Engineering Of The Protein Folding Pathway In The Endoplasmic Reticulum (ER) In Trichoderma Reesei And Study On The Expression Of Heterologous Protein BGLA And Lcc1

Filamentous fungi Trichoderma reesei is the main cellulase producer in industry,and its secreted protein can be over 40g/L.Besides,T.reesei is also an excellent host for heterologous protein production.However,The production of heterologous protein is usually lower than that of native protein.The bottleneck of this problem may occur in the folding and secreting process after protein translation.The endoplasmic reticulum(ER)is an important site for protein synthesis and maturation.After protein getting into ER,chaperone Bipl assists in protein folding,and disulfide bond isomerase Pdil as well as oxidoreductase Erol help to form the disulfide bond.Besides,the glycosylation status of secreted protein is also an important signal for detecting protein folding and sorting.Glucosidase Gls2 cuts off the glucose residue in the branch A of sugar chain.If the protein is folded correctly,it would transport out of ER.However,if it is folded incorrectly,mannosidase Mns1 would cut off the mannose residue in the branch B of sugar chain.Then the misfolded protein undergoes the ER-associated degradation pathway(ERAD),transport out of ER,and be degraded in the cytoplasm in the end.Or,one glucose residue is re-added in the branch A of sugar chain by the UDP-glucosyl transferase Gptl,the misfolded protein is refolded.The genetic engineering of related genes in ER protein folding pathway could promote protein folding and secretion in T.reesei,and thus constructe efficiently cellulase-producing strain.?-glucosidase(BGL)could hydrolyze cellobiose into glucose,and the insufficiency of BGL in the cellulase system is one of the bottlenecks affecting cellulose hydrolysis.It was founded that Aspergillus niger BGLA shows higher substrate specificity and specific enzyme activity than T.reesei Bgll.And BGLA exhibits less adsorption onto lignin,which indirectly facilitates enzymatic hydrolysis of cellulose.Laccase is a polycopper oxidase that could oxidize phenolic and aromatic amine substrates,which is mainly used for lignin degradation,pulp bleaching,contaminant detoxification,biological electrodes and so on.Besides,laccase are mainly found in basidiomycetes,and their commercial applications are hindered due to requiring toxic substances induction and the difficulty in culturing host strains as well as protein purification.Heterologous expression of laccase gene is one of the important ways to obtain high quality laccase.In this study,we constructed T.reesei bip1,pdi1,ero1,gpt1 and gls2 genes over-expressing strains and mnsl gene deletion strain.And the engineering of the protein folding pathway in ER was finished.Besides,the expression of A.niger?-glucosidase BGLA and Trametes trogii laccase Lccl were studied,using T.reesei as host.The specific research contents are as follows:1.The engineering of the protein folding pathway in ER was finished.We constructed chaperone Bip1,disulfide bond isomerase Pdil,oxidoreductase Ero1,glucosidase Gls2 and UDP-glucosyl transferase Gptl over-expressing strains as well as mannosidase Mns1 deletion strain.Fluorescence quantitative PCR showed over-expressing bip1,pdi1 and ero1 genes could cause UPR to some extent,but not activate ERAD;over-expressing gpt1 and gls2 genes as well as deleting mns1 gene could cause UPR and activate ERAD to some extent.When treated with DTT,the BGL activities of mutant strains were significantly higher than that of the parent strain.Under ER pressure,the extracellular BGL secretion ability of mutant strains were stronger than that of the parent strain.Besides,compared to the parent strain,the FPA and BGL activities of OEgpt1,OEero1,OEbip1 and Amns1 strains were remarkably improved.Over-expressing bip1 and pdi1,the FPA were improved 43.0%and 49.6%,respectively;BGL activities were enhanced 112.3%and 41.7%,respectively.The EG activities of OEgpt1,OEerol and OEbipl strains were a little higher that of the parent strain QP4.And The EG activities of ?mns1,OEgls2 and OEpdi1 strains were a little lower that of the parent strain.There were no significant changes in CBH activities of all the mutant strains.2.T.reesei successfully expressed A.niger ?-glucosidase BGLA.T.reesei inducible strong promoter Pcbhl was used to express A.niger BGLA.The generated strain T.reesei SCB18 exhibited a 29.8%increase in total cellulase activity and a 51.3-fold enhancement in BGL activity(up to 103.9 IU/mL).The over-expressing of A.niger BGLA caused UPR to some extent,but did not activate ERAD significantly.The A.niger BGLA required more chaperones Bipl and disulfide bond isomerase Pdil to complete the correct fold.We observed that the cellulase system of SCB18 showed signifcantly higher saccharifcation effciency toward diferently pretreated corncob residues than the parent strain SDC11.When the delignifed corncob residue was used as substrate,the cellulose conversion rate was 96.6%after a 96-h reaction;When the acid-pretreated corncob residue was used as substrate,the cellulose conversion rate was 53.1%after a 96-h reaction.Moreover,the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize cellobiose,gentibiose and sophorose from glucose.The biggest glucose conversion rate was achieved after a 2-d reaction using 60%glucose as substrate under 65 ?.The transglycosylation product was finally utilized as the inducer for cellulase production,which provided a 63.0%increase in total cellulase activity compared to the frequently used soluble inducer,lactose,thus representing an alternative and prospective candidate as carbon source for larger production of economically attractive and competitive cellulase.3.The study on the expression of T.trogii laccase Lccl that having high oxidation-reduction potential by T.reesei.The constitutive strong promoter Pcdnal was used to express T,trogii Lccl,and 7 transformates were obtained.T.trogii laccase lccI gene was successfully transcribed in T.reesei,but no activity was detected according to the results of fermentation culture and ABTS chromogenic analysis.The over-expressing of T.trogii Lccl caused UPR and activated ERAD strongly.Laccase Lccl may not be folded correctly in T.reesei ER,then was transported out of ER,and be degraded by proteasome in the end.4.Pichia pastoris GS115 successfully expressed laccase Lccl and its glycosylated mutant proteins.No mutation,single mutation and double mutation were performed on 54 and 433 site glycosylation sites of laccase Lccl,respectively.And the non-mutant Lccl strain,single-mutant N54Q strain,single-mutant N433Q strain and double-mutant NDQ strain were obtained.The laccase activities were as that:Lccl>N54Q>N433Q>NDQ.The results of laccase purification and activity detection showed that the glycosylation of 433 asparagine was essential for laccase Lccl to maintain activity.The both optimum pH of Lccl using ABTS and 2,6-DMP were 5,and the optimum temperatures of Lccl using ABTS and 2,6-DMP were 60? and 55 ?,respectively.