Study On Regulation Of Secondary Metabolism In Aspergillus Niger FGSC A1279

Filamentous fungi are producers of secondary metabolites (SM) that include the commonlyused antibiotics penicillin, anticholesterol drug lovastatin and harmful toxins such as aflatoxinand sterigmatocystin. Aspergillus, as a kind of filamentous fungi, plays vital role in industry.In particular, Aspergillus niger (GRAS strain) is recognized as an important producer ofenzymes and organic acids. The complete genome sequences of A. niger became availableand this has led to the identification of a large number of secondary metabolic genes. Thisindicates that A. niger has the potential to produce a number of secondary metabolites.However, the regulatory factor and mechanism remain to be investigated. Due to the avobereason, A. niger FGSC A1279was selected for further study. The contents and results arelisted as following.1. Whole genome sequence of A. niger FGSC A1279Genome sequencing of A. niger FGSC A1279was conducted by paired-end sequencingstrategy on the Illumina high-throughput sequencing platform. The assembled genome ofFGSC A1279was35.45Mb with a sequencing depth of134fold including336contigs(>400bp). There were11686ORFs distributed in the eight chromosomes based on theannotated information. Syntenic analysis between A. niger FGSC A1279and CBS513.88indicated that backbone genes in SM clusters were similar, however, genes involved in SMclusters were different.2. Construction and metabolic profile studies of mutants related with SMHistone methylation, acetylation and deacetylation gene deletion mutants and globalregulatory factor laeA, type III polyketide synthase (AnPKSIII) and sclerotia growthregulatory factor AnsclR deletion and overexpression mutants were constructed. Metabolicprofile study showed that metabolites of histone modification deletioin, laeA, AnPKSIII andAnsclR overexpression strains were altered, of wihich spectrum of laeA overexpression strainwas greater influenced.3. Transcriptome studies of laeA deletion and overexpression staiansRNA samples of A. niger laeA deletion and overexpression staians were sequenced by RNA-Seq technology and genome-wide transcriptome profile was obtained. The genestructures and alternative splicing (AS) events of A. niger were analyzed based on RNA-Seqdata. The results showed that7387extended5’UTRs and74693’UTRs of transcribed geneswere predicted;3266uORFs were detected in the identified5’UTRs. Four kinds of AS eventsincluding retained introns, skipped exon, alternative5’splice site and alternative3’splice sitewere detected.Whole-genome comparison of the transcriptional profile analysis showed that expressionlevel of3673gene were upregulated in laeA overexpressioin strain compared to the laeAdeletion strain. ClueGO analysis showed that the upregulated genes were mainly distributedin biosynthetic pathways of secondary metabolites and regulation of rRNA processing andprotein translation. The results also indicated that27.63%(286of1035genes) of genes in SMclusters were upregulated including28backbone genes (16PKSs,9NRPSs,2Hybrids,1DMAT) in laeA overexpression strain compared to laeA deletion strain.4. Isolation, identification and studies of other influencing factors of metabolitesThe metabolites were purified through silica gel, ODS and glucan (Sephadex LH-20)column combined with HPLC and structure of the compounds were indentified by LC-MS,NMR and IR. The results showed that metabolites of laeA, AnPKSIII and AnsclRoverexpression strains including p-hydroxybenzoic acid,3,4-dihydroxy benzoic acid,diisobutyl phthalate and a steroid analogs were indentified. These data built foundation for theresearch of A. niger secondary metabolites.Metabolite analysis of AnPKSIII overexpression strain indicated that its biosyntheticpathway might be similar to that of3,5-dihydroxybenzoic acid in A. oryzae catalyzed by typeIII polyketide synthase csyA; results also showed that sclerotia regulatory factor AnsclR andits metabolites were associated with oxidative stress.