Heterologous Expression Of Cellobiohydrolase Te-Cel7A In Trichoderma Reesei And Study Of The Gene Function Of Serine Protease Spt1

Trichoderma reesei is an industrially important filamentous fungus that produces cellulolytic enzymes for biomass conversion.However,the process of industrial degradation of lignocellulose usually requires the properties of cellulase with thermal stability.Therefore,the expression of thermalstable cellulase in T.reesei is one of the strategies to improve the cellulase system.In addition,the protease in T.reesei may affect the phenotype of the strain and the expression of protein secretion,but the understanding of it is not clear at present.Here,the thermalstable cellobiohydrolase Te-Cel7 A was heterologous expressed in T.reesei to optimize the cellulase system.Furthermore,the gene function of serine protease Spt1 was analyed.Finally,the Spt1 was over-expressed in the engineering strain that have thermalstable cellulase heterologous expression.The main contents are described as follows:(1)In this study,the constitutive promoter cdna1 and CBH1-SP was used to construct the Te-Cel7 A expression cassette.Then,the Te-Cel7 A expression cassette was transformed into the QP4 protoplasts.The transformants were screened on AMM plates.One of the transformants,namely QTC14,showed the clearest cellulolytic halo around the colony.The transformant QTC14 could successfully secrete the recombinant Te-Cel7 A into the supernatants using glucose as carbon source.The enzyme had temperature optima around 65 ? and an optimal pH of 5.0,which are in accordance to those from the native host.In the fermentation condition for cellulase production,QTC14 exhibited a 28.8% increase in cellobiohydrolase(CBH)activity and a 65.0% increase in filter paper activity(FPA).In addition,the QTC14 cellulase system showed higher thermal stability for both CBH and FPA than that of the parental strain QP4.When the recombinant cellulase system was used for saccharifcation of lignocellulosic materials.It was found that the QTC14 cellulase showed higher saccharification efficiency towards differently pretreated corncob residues than the QP4 cellulase.Especially,in the saccharification of delignified corncob residues,the cellulose conversion of QTC14(49.3%)showed 13.9% higher than that of QP4 after 48 h reaction.These results reveal that the thermophilic fungusderived cellulases could be efficiently expressed and the resulting recombinant cellulases have potential applications for biomass conversion.(2)The protein sequence analysis of the serine protease Spt1 of T.reesei showed that the Spt1 belongs to subtilisin-like protease in the subtilases family,which has the leading peptide and may belong to the vacuole protein.To explore the function of Spt1 of T.reesei,we constructed Spt1 deletion strain?spt1 and complementation Rspt1 respectively.Deletion of spt1 made the sporulation impaired.When the spt1 was introduced into the T.reesei ?spt1,the sporulation process could be rescued.This indicates that Spt1 is related to the phenotypic stability of T.reesei.At the same time,deletion of spt1 was not affected the growth rate of ?spt1 on different carbon sources,but it affected the cellulose degradation ability of the strain.By analyzing the extracellular cellulase activity of ?spt1 and Rspt1,the extracellular protein secretion,FPA,EG activity,CBH activity and BGL activity of ?spt1 decreased significantly.When the spt1 was introduced into the T.reesei ?spt1,the cellulase activity could be rescued.It showed that spt1 is related to the cellulase synthesis and secretion of T.reesei.In order to confirm whether Spt1 is located in the vacuole,the cellular location of Spt1 is further studied by using green fluorescent protein.The results showed that Spt1 was located in the vacuole.In order to study the effect of Spt1 on the optimization of cellulase system,we constructed Spt1 overexpressing strain SOE and SOD by using spt1 promoter and constitutive promoter cdna1,respectively.By analyzing the cellulase activity of SOE and SOD,the overexpression of Spt1 could significantly improve the cellulase activity of T.reesei.Especially for Spt1 overexpressing strain SOD,which was constructed by using a strong promoter cdna1,its extracellular protein content,FPA,EG activity,CBH activity and BGL activity increased by 65.9%?140%?48.8%?265% and 260% compared with the QP4 strain.It is suggested that spt1 is an effective target for improving the enzyme system of T.reesei.(3)In order to further optimize the cellulase system of T.reesei.We overexpressed Spt1 in T.reesei QTC14,which heterologous expressed the thermalstable cellobiohydrolase Te-Cel7 A in T.reesei,and successfully constructed T.reesei STC.By analyzing cellulase activity of STC,QTC14 and QP4,it was found that the extracellular protein content,FPA,EG activity,CBH activity and BGL activity of STC increased by 10%,10%,22%,15%,34%,respectively,compared with QTC14 enzyme activity.The results showed that through the overexpression of SPT1 in T.reesei QTC14,the cellulase system of T.reesei QTC14 could be further improved,thus contribute to reduce the production cost of cellulase and promote the development of bioethanol industry.