Identification Of P450 And BZIP Genes In Ochratoxin A Biosynthesis Pathway In Aspergillus Niger

In recent years,Ochratoxin A(OTA)produced by Aspergillus niger was potentially harmful to human health.The study found that OTA synthesis required a series of enzymatic reactions,and the genes that encoded these enzymes were clustered to clusters.Based on bioinformatics analysis,bZIP(Gene ID:4988390)and P450(Gene ID:4988391)might be genes within clusters in the OTA biosynthetic in A.niger,but was not knocked out and verified.A.niger A14 produced OTA was used as the initial strain in this study.A.niger gene disruption strains and overexpression strain were constructed by Agrobacterium-mediated homologous recombination.The role of bZIP and P450 in A.niger infection and OTA synthesis was verified,which might provide theoretical insight towards effective control of the A.niger toxin-producing and further exploration of the OTA in network control.A14?bZIP and A14?P450 strains were selected by Agrobacterium infection.The positions of the HYG gene insertion were determined by genomic re-sequencing in chromosome An15 of A.niger CBS 513.88 1,857,026-1,859,864 and 1,859,284-1,862,245 by 1 gene copy reverse insert,respectively.The results showed that the A14?bZIP and A14?P450 strains had the stable inheritance.Overexpressed bZIP gene strain A14::bZIP was selected by Agrobacterium infection.The results showed that the A14::bZIP strain had the stable inheritance.Compared with A.niger A14 phenotype,there were no any significant differences at growth rate,colony morphology,conidial development and biomass of A14?bZIP,A14?P450 and A14::bZIP strains.In this study,OTP,OTa,and OTA could not be produced during the 9 days culture in A14?bZIP strain.However,in A14::bZIP strain,those production increased to different degrees.This result confirmed that the gene bZIP was involved in the biosynthetic pathway of OTA.There was delayed and decreased OTa and OTA production after the P450 gene was destroyed,but the production of OT? was significantly increased.This result confirmed that the P450 gene was involved in the biosynthetic pathway of OTA and it might catalyze the cross-linking of the phenylalanine and the dihydroisocoumarin.Gene expression was analyzed by qRT-PCR,the results showed that the deletion of bZIP gene reduced the expression levels of pks,nrps and p450 in different degrees.Moreover,the expression levels of pks,nrps,p450 and hal genes in the bZIP overexpressed strain were higher than those in the wild strain.It was further proved that the bZIP gene was the intra-cluster regulator in the OTA biosynthetic gene cluster.