Study On Feedback Regulation Of Secretion Stress In Aspergillus Oryzae

Aspergillus oryzae can produce large quantities of proteins that making it a suitable host for protein production.Compared to the large amounts of homologous proteins produced by A.oryzae,the production of heterologous proteins is often limited.Inefficient protein transit,sorting,and secretion of heterologous proteins result in the accumulation of unfolded proteins in endoplasmic reticulum(ER).When protein folding requirements exceed the ER's folding capabilities,unfolded proteins can accumulate and elicit stress to the ER,which will activate the unfolded protein response(UPR)mechanism to decrease ER stress.In filamentous fungi,the UPR mainly depends on an evolutionarily conserved signaling cascade that is mediated by the ER-resident transmembrane kinase/endoribonuclease IRE1 and the basic leucine zipper(bZIP)transcription factor HacA.In A.oryzae,the majority of these studies induced the UPR signaling pathway through the use of harsh chemicals(dithiothreitol or tunicamycin),homologous or heterologous protein expression,or growth conditions that induced secretory hydrolytic enzyme production.However,HacA was used only as an indicator of UPR process;thus,the specific contributions of HacA could not be evaluated.In filamentous fungi,beside the conventional UPR response toward ER stress,exist another significant protective mechanisms to decrease the protein load of the ER,termed repression under secretion stress(RESS).Although the exact mechanism by which RESS operates is unknown,it has been shown to take place at the transcriptional level,and to be mediated through the promoter of the gene involved.However,the cis-element of the secretory protein promoters that is required for RESS is still unknown.In this study,to comprehensively evaluate the role of HacA in Aspergillus oryzae,the inactivation and constitutive expression of UPR mutants were successfully generated,and transcriptome analyses of HacA-DE and HacA-CA mutants were also performed.Furthermore,to investigate the RESS mechanism,we analyzed the expression of the Taka-amylase A gene(amyB)in A.oryzae,which was depressed under secreted protein stress.The detailed results of this thesis are listed below.1.Establishment of highly efficient gene-targeting recipient strains for advanced gene analyses The highly efficient gene-targeting system(niaD-;ku70::ptrA)was generated by disruption of the gene encoding Ku70 that play a role in nonhomologous chromosomal integration in A.oryzae.Futhermore,establishment of multiple gene disruptions recipient strain(niaD-;ApyrG;ku70::ptrA)by disruption of the pyrG gene for uridine/uracil auxotroph with highly efficient gene-targeting background(Aku70).We further performed prtT disruption(AprtT)by the pyrG marker with high gene-targeting efficiency allowed by the Aku70 background.After disruption process the pyrG marker was excised by the direct repeats consisting of 500 bp upstream flanking region of the target gene,resulting in no residual ectopic/foreign DNA fragments in the genome(AprtT-RE).Moreover,deletion of A.oryzae prtT resulted in the loss of secreted protease activity.The expression of three secreted proteases(pepB,pepD and Alkaline protease)was markedly reduced.2.Effects of inactivation and constitutive expression of UPR on vegetative growth and secreted protein production The homokaryotic hacA disruption mutants(HacA-DE,HacA-DE-RE),and a series of constitutively active UPR mutants(HA(HacA-CA),HA-gpdA,HA-amyB,HAGFP,HALGFP,HAGFP-gpdA,HALGFP-gpdA,HAGFP-amyB,HALGFP-amyB)were successfully generated in A.oryzae.Growth and phenotypic profiles demonstrated that hyphal growth and sporulation were impaired in all inactivation and constitutive expression of UPR mutants.Additionally,the SDS-PAGE analysis of culture supernatants revealed that secreted protein levels were decreased also in all mutants.Hence,We assume that HacA must maintain a threshold activity level,or deleterious effects,such as inhibition of cell growth and protein secretion,will occur.3.Transcriptomic analysis of the key unfolded protein response transcription factor HacA The combination of a genetically defined,constitutively activated HacA mutant and a hacA disruption mutant have provided a solid basis for a genome-wide expression analysis to study the response of A.oryzae toward ER stress.The results indicate that the differentially expressed genes in these strains are mainly involved in the protein secretory pathway,amino acid metabolism,lipid metabolism,and carbohydrate metabolism.We identified 80 and 36 genes in the secretory pathway,which mostly belonged to protein folding/UPR,glycosylation,and vesicle transport processes,whose expression was significantly changed in the HacA-CA strain compared with the WT and HacA-DE strains,and in the HacA-DE strain compared with the WT strain,respectively.Additionally,we compared the transcriptome of the HacA-CA strain with four other relevant transcriptomes of A.oryzae.Overall,28 genes were found to have either elevated(19 genes)or lowered(nine genes)transcript levels under all conditions that were examined,thus defining the core set of genes that are important for ensuring high protein traffic through the secretory pathway.Furthermore,the constitutive expression of activated HacA and the disruption hacA both exhibited reduced expression of extracellular enzymes,especially amylolytic enzymes,which resulted from the activation of the repression under secretion stress mechanism in response to endoplasmic reticulum stress.4.Identification of functional cis-elements required for repression of the Taka-amylase A gene under secretion stressTo investigate the RESS mechanism,we analyzed the expression of the Taka-amylase A gene(amyB)in A.oryzae,which was depressed under secreted protein stress.We conducted a truncation and deletion analysis of the amyB promoter to identify cis-elements required for RESS.A nucleotide sequence(positions-378 to-291)without any binding sites for the transcriptional activator AmyR,which is involved in amylolytic gene expression,was required for RESS.The octamer sequence TCACGGGC(positions-307 to-300)constituted the core sequence of the upstream activating element essential for amyB down-regulation under secretion stress.Both the inactivation of AmyR and RESS contributed to the down-regulation of amyB expression under ER stress.