Function Analysis Of The Genes Involved In Protein Degradation In Aspergillus Niger

Filamentous fungi can secrete large amounts of proteins.They hold many characteristics such as high secretory capability and post-translational modifications similar to that of mammal.With the advances in fungal molecular genetics,strain improvement,and especially fungal genomics, filamentous fungi are developed as microbial cell factories for the production of homologous and heterologous proteins.However for production of heterologous proteins,the yields are still poor. Protein degradation and low secretion efficiency are the major reasons.To control protein degradation thus to improve expression of heterologous protein,two protease genes apsB,ptrB were studied to be disrupted through gene targeting to further study the influence of their disruption on expression of homologous an heterologous protein in this dissertation.The apsB gene encodes one type of lysine aminopeptidases which release specific lysine residue from the N-terminal of polypeptides.The apsB gene was also found highly identities in Neosartorya fischeri,Aspergillus fumigatus,Aspergillus clavatus,Aspergillus oryzae,Aspergillus terreus, Aspergillus nidulans,and so on.So far,we don't know about any of its biochemical characteristics nor its function in Aspergillus niger.The ptrB gene encodes one type of membrane transport proteins that import dipeptides and tripeptides.It also was found in A.fumigatus and A.nidulans.Firstly,DNA fragments of corresponding genes from A.niger GICC2773 were amplified using the primers designed according to the sequences of apsB and ptrB gene and ligated with cloning vector pBS-T or pUCm-T.Then hph or amdS expression unit were inserted into the middle part of each gene to generate respective gene disruption plasmids pBSΔapsB-amdS and pUCmΔptrB-hph.Secondly,the disruption plasmids pBSΔapsB-amdS and pUCmΔptrB-hph were digested with respective corresponding restriction enzymes and transformed into A.niger GICC2773 strain in which a laccase expression vector had been integrated.The generating transformants were screened for disruption strain using PCR method.The results showed thatΔapsB#28,ΔapsB#93 andΔptrB#33 were disruption strains of apsB and ptrB gene,respectively.In addition,However, the disruption plasmid pBSΔapsB-amdS was transformed into A.niger A12,but no apsB disruption strain could be found whereas over total 500 transformants were screened.At last,glucoamylase/a-amylase and laccase activity were analyzed in these disruption strains to study the influence of each gene disruption on expression of homologous protein and heterologous protein.The data of enzyme assay showedΔapsB#28,ΔapsB#93 improved expression of homologous protein Glucoamylase by 77-85.2%,36.6-47%,respectively,and improved heterologous protein laccase by 45-59.2%,34-39.2%,respectively.InΔptrB#33,the laccase activity was improved by 5.3%,a-amylase activity had no influence.All value in this paper were counted with average level.