Study On The Relationship Between Aspergillus Oryzae Protease And Heterologous Protein Expression Level

Aspergillus oryzae,a filamentous fungus,is a safe strain widely used in traditional food fermentation and a mature platform for producing enzyme preparations.Based on the strong protein production and secretion ability of Aspergillus oryzae and the mature genetic transformation operation,it has great potential to be transformed into a chassis strain that produces heterologous proteins,especially complex glycoproteins.An important factor in limiting heterologous protein expression in Aspergillus oryzae is the large number of proteases secreted by itself.These proteases degrade heterologous proteins extracellularly,while also competing with heterologous proteins for intracellular pathways.In this paper,starting from 14 strains of Aspergillus oryzae used in different fields,a method for extracting secreted proteins from soft agar medium was first established.The protease activity of these strains was determined using the folin phenol reagent.Protease production capacity was defined as protease activity per unit weight of Aspergillus oryzae.The 14 strains were divided into three groups with high protease activity,medium protease activity and low protease activity according to the above criteria.Four strains of three groups of enzyme activity were selected for RNA-seq and analysis.Finally,274 differentially expressed genes were screened,and further analysis found that 2 upregulated expression genes were directly annotated as protease-coding genes.After the enrichment analysis,it was found that the difference in protease activity of different strains was also related to the secretion capacity and growth state of the protein.Then,Aspergillus oryzae strain transferred into p EX2B-amy B-FSH vector was used to try to express the heterologous protein follicle-stimulating hormone(FSH).After comparing the wild-type strain and the transformed strain,it was found that the heterologous protein FSH could not be detected both intracellular and extracellular of the transformed strain.Starting from the protease activity,this paper explores the shortcomings of Aspergillus oryzae as a heterologous protein expression platform,and points out the direction for its further transformation by using transcriptome sequencing and analysis.At the same time,in the process of trying to express heterologous glycoprotein FSH by using the strategy of fusion expression,the difference of protein secretion ability between strain 2035 and 3042 was further verified.In the selection of heterologous protein expression strains of Aspergillus oryzae,the strains with lower protease activity should be selected on the premise of ensuring the secretion efficiency.The differentially expressed genes screened in this study can provide a reference for the evaluation of protease activity of different strains.