Functional Analysis Of The Genes Involved In Secretion Of Heterologous Protein In Aspergillus Niger

Filamentous fungi are widely used in production of eukaryotic protein due to their following characteristics: high secretory capability, post-translational modifications similar to that of mammal and so on. However for production of heterologous proteins, the yields are still poor. Proteolytic degradation and low secretion efficiency are the major reasons. To control proteolytic degradation thus to improve expression of heterologous protein, three protease genes dppIV, dppV and pepAc were studyed to be disrupted through gene targeting to further study the influence of their disruption on expression of heterologous protein in this dissertation. The dppIV and dppV genes encode two types of dipeptidyl peptidases which release specific dipeptides from the N-termini of polypeptides. It was reported that DPPIV and DPPV participated in degradation of extracellular protein in A. oryzae and A. fumigatus. The pepAc gene was found to be a homolog of pepA gene and was identified newly from the genomic sequences of A.niger. This dissertation tried to study whether PEPAC was also one of the extracellular acid proteases involved in proteolytic degradation of heterologous protein. In addition, through gene targeting the mnn10 gene which encoded one type of mannosyltransferase was disrupted in order to study the influence of its disruption on expression of heterologous protein and the function of mnn10 gene in biosynthesis of high mannan chain of glucoprotein in A.niger.DNA fragments of corresponding genes were amplified using the primers designed according to the sequences of dppIV, dppV, pepAc and mnn10 gene and ligated with cloning vector pBS-T or pMW1.Then hph expression unit or amdS gene were inserted into the middle part of each gene to generate respective gene disruption plasmids pBSΔdppIV-hph, pBSΔdppIV-amd, pBSΔdppV-hph, pBSApepAc-hph and pMW1Δmnn10.The disruption plasmids pBSAdppIV-hph, pBSAdppV-hph, pBSApepAc-hph and pMWIAmnn10 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 that AdppIV#17, AdppV#26 and Amnn10#2 were disruption strains of dppIV, dppV and mnn10 gene, respectively. However, no pepAc disruption strain could be found whereas total 286 transformants from transformion of pBSApepAc-hph were screened.Using similar method, pBSAdppIV-amd was transformed into dppV disruption strain AdppV#26 and the dppIV and dppV double disruption strain AdppVAdppIV#2-3 was achieved.Laccase activity were analyzed in these disruption strains to study the influence of each gene disruption on expression of heterologous protein. The data of enzyme assay showed disruption of dppIV , dppV and mnn10 gene improved expression of heterologous protein laccase by 8.3% in ΔdppIV#17, by 17.4% in ΔdppV#26 and by 13.8% in Δmnn10#2. The double disruption of dppIVand dppV further improved expression of heterologous protein laccase by 22.4 % in AdppVAdppIV#2-3.