Construction Of The Protein Expression And Secretion System In Aspergillus Niger ATCC 20611 Using The Promoter Pcdna1 From Trichoderma Reesei

The filamentous fungus Aspergillus niger has become an excellent system for the expression of heterologous proteins,due to its efficient protein translation,folding and post-translational modification,strong capacity for protein secretion,good safety and mature fermentation process.However,the high secretion of endogenous proteins in A.niger not only impedes the continuous and efficient production of heterologous proteins,but also reduces the purity,which has become the bottleneck for the further development and application of A.niger protein expression system.A.niger ATCC 20611 is an industrial fructooligosaccharide-producing strain.It not only has the general advantages of the A.niger in the expression of heterologous proteins,but also has the pure extracellular protein secretion background because the major enzyme ?-fructofuranosidase(FopA)produced by it,is anchoring to the cell wall.A.niger ATCC 20611 is expected to serve as a new host for the expression of heterologous proteins with a low endogenous protein secretion background,and then develop into an excellent protein expression and secretion system of A.niger.In this paper,based on the low endogenous protein secretion background,A.niger ATCC 20611 was selected as the research object to establish the system of heterologous protein expression and secretion.At first,by comparing the expression levels of the model protein driven by the commonly used strong promoter glycerol triphosphate dehydrogenase Pgpd1 in the glycolytic pathway of Trichoderma reesei and the strong promoter Pcdnal of T.reesei,the latter(promoter Pcdna1 of T.reesei)was selected to drive the efficient expression and secretion of the heterologous protein in A.niger ATCC 20611.Then,the promoter Pcdnal was further applied to express the capsid protein VP60 of the rabbit hemorrhagic disease virus(RHDV)in A.niger ATCC 20611.Finally,The promoter Pcdna1 was combined with the cell wall-anchoring domain FopA-C to realize the cell wall localization of the ?-galactosidase and glutamate decarboxylase in A.niger ATCC 20611.The details are as follows:1.Comparison of the expression levels of the model protein driven by the promoter Pgpd1/Pcdna1 from T.reesei in A.niger ATCC 20611.Firstly,in order to detect the availability of the conserved heterologous promoter Pgpd1 in A.niger and the feasibility of A.niger ATCC 20611 as the host for heterologous protein expression,the conserved glyceraldehyde-3-phosphate dehydrogenase promoter Pgpd1 from T.reesei was selected to drived the expression of model proteins,the green fluorescent protein EGFP and ?-glucosidase BGLA.The results observed under the fluorescence microscope showed that the promoter Pgpd1 of T.reesei could successfully drive the expression of EGFP.The results of esculin plate showed that the transformants had obvious halos,which proved the ?-glucosidase BGLA was successfully expressed.The highest enzyme activity of the transformant was 1.0 U/mL.These results indicated that the promoter of T.reesei could drive the heterologous protein expression in A.niger ATCC 20611.Then,the unique constitutive promoter Pcdna1 of T.reesei,which was stronger than Pgpd1,was selected for protein expression in A.niger ATCC 20611.The cassette for expression of secreted protein BGLA driven by Pcdnal was constructed and transformed into A.niger ATCC 20611.The transformants all had obvious halos on the esculin plate,and the ?-glucosidase activity was significantly increased.The highest ?-glucosidase activity of the transformants reached 15.2 U/mL.which was 14.2 times higher than that of BGLA expressed by Pgpd1.At the same time,SDS-PAGE results performed that the band of BGLA was single and clearly visible.Morever,in the total extracellular protein of transformants,BGLA accounts for more than 80%,which suggested that the heterologous protein can be expressed efficiently and with high purity by using Pcdna1 in A.niger ATCC 20611.2.Expression of the RHDV capsid protein VP60 driven by Pcdnal from T.reesei in A niger ATCC 20611.Firstly,the strong promoter Pcdna1 of T.reesei was used to directly drive the expression of VP60.It was found that VP60 band was invisible by protein electrophoresis under the condition that the fermentation supernatant was not concentrated.Then,through the further concentration of fermentation broth and mass spectrometry,it was confirmed that VP60 was successfully expressed,but the expression level was low.In order to improve the secretion of VP60,the three carriers containing endoglucanase EGII,chitinase CHI46 and ?-glucosidase BGLA,which are easy to detect the expression level,were selected for fusion expression with VP60.At the same time,considering the influence of different positions of carriers on the secretion,six different expression cassettes were constructed.The expression of carriers were successfully detected in the corresponding plates and the fermentation supernatants,which provided strong support for the successful expression and secretion of the heterologous protein VP60 in filamentous fungi.3.Construction of strains expressing cell wall-anchoring ?-galactosidase and glutamate decarboxylase.Since the strong promoter Pcdnal of T.reesei can drive the expression and secretion of genes in A.niger ATCC 20611 at a high level,in this paper,it was combined with the cell wall-anchoring domain FopA-C to construct a new localization expression system to express the cell wall-anchoring ?-galactosidase and glutamate decarboxylase.On the one hand,?-galactosidase was localized and expressed by the strong promoter Pcdna1 of T.reesei.The expression cassette GalV-FC containing the two-site mutant ?-galactosidase GalA-V and FopA-C was used as the experimental group.The expression cassette of Gal-FC containing the ?-galactosidase GalA and FopA-C and the expression cassette of GalV-MP containing the two-site mutant ?-galactosidase GalA-V and FopA-C were used as the control groups.Then,the above expression cassettes were transformed into A.niger ATCC 20611 respectively.So far,the purified transformants of each cassette have been successfully obtained by PCR verification.On the other hand,glutamate decarboxylase was localized and expressed by the strong promoter Pcdnal of T.reesei.Glutamate decarboxylase from Escherichia coli,Lactobacillus brevis,Saccharomyces cereviscera and A.niger were fused with FopA-C to construct the expression cassettes GADEc,GADLb,GADSc and GADAn.At present,the transformants have been verified by PCR and purified.This chapater laid the foundation for the localization of ?-galactosidase and glutamate decarboxylase in A.niger ATCC 20611.