Development Of A Protein Secretion System With Low-protease Background And Functional Analysis Of The Protein Complex That Guards Folding Polypeptides In Trichoderma Reesei

The filamentous fungus Trichoderma reesei has been used for protein production due to its high natural capacity of extracellular enzyme secretion.Nonetheless,the yields of heterologous proteins using T.reesei as a host is unsatisfactory,which is mainly due to the unwanted degradation of target proteins caused by extracellular proteases.Therefore,it is necessary to investigate the regulatory profile of extracellular protease secretion and to construct an extracellular protease deficient strain in T.reesei.Knockout of the transcriptional factor involved in the extracellular protease secretion could be an efficient strategy to achieve extracellular protease reduction.According to the related reports and combined with the findings in our lab,it was found that the p53-like transcriptional factor Vibl participates in the regulation of both extracellular proteases and cellulases in T.reesei.The deletion of the vibl gene could result in the dramatically reduced extracellular protease activity and severely compromised cellulase activity.Thus,the vibl deletion strain exhibits low-protease and cellulase-free background,suggesting that it has a great potential for heterologous protein production.Besides this,the difficulty of heterologous protein folding is another non-negligible limitation for it to achieve high production.The guardian factor Slpl-Emp65 complex(SE complex)can protect folding polypeptides from degradation,which is a potential target to increase the efficiency of protein folding.To reduce the extracellular protease production and enhance the heterologous protein folding,the profile of extracellular protease secretion by T.reesei was investigated Moreover,the development of a novel protein expression system and functional analysis of SE complex were carried out in this study.The specific research contents of this study are as follows:1.The regulatory profile of extracellular proteases was investigated.The effects of carbon(glucose and lactose)and nitrogen sources(ammonium sulphate,sodium nitrate,peptone and corn steep liquor)were analyzed through the skim-milk agar plates and protease activity determination.It was found that nitrogen sources could regulate the extracellular protease production.Organic nitrogen sources were beneficial for protease production,whereas the preferred nitrogen source ammonium sulphate was an inhibitory factor.In addition,lactose was a more effective factor than glucose for extracellular protease secretion.Moreover,the protease activity decreased by more than 70%when serine protease inhibitor PMSF was added to fermentation supernatant;and the proteases activity was slightly inhibited by metalloprotease inhibitor EDTA.These results indicate that serine proteases are responsible for the main part of the extracellular protease activity and metalloproteases are also present.Furthermore,29 extracellular proteases were identified by LC-MS/MS,including 13 serine proteases,6 aspartic proteases and 10 metalloproteases.Specifically,7 extracellular proteases were present among all conditions tested in this study.2.A novel protein secretion system was developed in this study.The vib1 deletion strain(?vib1)exhibited the dramatic decrease in both cellulase and protease production,whereas the growth of ?vib1 was comparable to that of the parental strain QM53,indicating that ?vib1 has a great potential for heterologous protein production.Therefore,the Aspergillus niger ?-glucosidase coding gene bglA using the cbh1 signal peptide and the cdnal promotor was expressed in ?vib1 and QM53 to demonstrate its feasibility as a host for protein production.It was further found that the transcriptional levels of the genes involved in the unfolded protein response(UPR)were decreased in?vib1 compared with the parental strain QM53 by RT-qPCR analysis,including bipl and pdi1.3.The function of SE complex was preliminarily analyzed.The SE complex mutant strains were constructed by homologous recombination,including slp1deletion strain(?slp1),slp1 overexpression strain(OE-slp1),emp65 deletion strain(?emp65),emp65 overexpression strain(OE-emp65),slp1 and emp65 deletion strain(?SE)and slp1 and emp65 overexpression strain(OE-SE).The growth rates of the above mutants were measured under the condition containing 10 mm DTT.It was found that the growth of ?emp65 became slower,indicating that the sensitivity of ?emp65 to DTT was increased.And the ?SE strain could not grow on this medium with 10 mm DTT.In addition,the FPA(filter paper activity)of ?SE strain was decreased by about 25%and the CBH(cellobiohydrolase)activity reduced by approximately 50%.It was also showed that the deletion or overexpression of the slp1 gene could induce the up-regulation of derl.In order to further determine whether SE complex was involved in the process of polypeptide folding,ER-GFPfast was expressed in the parental strain and the ?slp1 strain.Moreover,the fluorescence intensity of ER-GFPfast secreted in the ?slp1 strain was lower than that of QM53.