Mechanisms Of Epigenetic Regulation On Secondary Metabolites In Aspergillus Niger

Secondary metabolites can be of great value in the medical field as antibiotics and immunosuppressants.With the increase of bacterial resistance and the emergence of superbugs,the search for new antibacterial substances has become a common concern for many researchers.The genomic information of Aspergillus niger strain CBS513.88 showed that the strain possesses dozens of secondary metabolite gene clusters,indicating that Aspergillus niger has the potential to synthesize a variety of secondary metabolites.However,there are few reports on secondary metabolites in Aspergillus,mainly because the secondary metabolism synthesis gene clusters are generally silenced under laboratory conditions,so different methods need to be found to activate the expression of silenced genes.In this thesis,the effects of histone acetylation and deacetylation modifications on secondary metabolism were studied in Aspergillus niger FGSC A1279,and the specific regulation of secondary metabolic gene targeting was achieved using CRISPR/d Cas9 technology.The details and results are as follows.(1)Crude extracted metabolites of Aspergillus niger FGSC A1279,Δgcn E and gcn E-com(complementation strain)were analyzed by LC-MS and 11 known compounds were identified.Six compounds were purified by semi-preparative HPLC,one of which was not reported and was named nigerpyrone,and five were reported substances.The six substances were analyzed using high resolution electrospray mass spectrometry(HR-ESIMS)and 1D/2D nuclear magnetic resonance(NMR)to determine their compound structural formulae and to predict the biosynthetic gene clusters for the compounds Funalenone(4),Fumonisin B2/ B4,IsokotaninA.The synthetic gene clusters of the substances nigerpyrone(1),carbonarone A(2)and pestalamide A(3)were identified by knockout and biosynthetic models for the three substances were proposed.(2)The eight histone deacetylase genes of Aspergillus niger FGSC A1279 were knocked out separately using homologous recombination and seven knockout strains were successfully obtained.The seven successful knockout strains and the original strains were cultured on different media to analyze the effects of osmotic pressure,temperature,oxidative stress and chemicals on the mutant strains.hos A deletion was found to have a significant effect on the phenotype and transcriptome analysis was performed for Δhos A and Δhda A.GO functional annotation of the differential genes indicated that hos A and hda A had different degrees of effect on secondary metabolism,pigment synthesis,and cell wall formation.Changes in secondary metabolites were examined using LC-MS,which showed a significant reduction in Fumonisin B1/B2 synthesis after hos A or hda A knockout.(3)The effect of gcn E and hos A gene deletion on chromatin openness in Aspergillus niger FGSC A1279 was investigated using ATAC-seq.The results showed that about one thousand specific chromatin open regions existed in each of the Δgcn E and Δhos A strains compared to A.niger FGSC A1279,indicating that knockdown of the genes gcn E or hos A affects chromatin openness.The open regions were mainly concentrated near the transcription start site(TSS),with more than 90% of peaks concentrated within 1 kb upstream of the ATG.Functional annotation of the differential gene GO indicates that gcn E and hos A deletions primarily affect transcriptional regulation.Combining RNA-seq and IGV visualisation to investigate the relationship between chromatin openness and transcription levels of secondary metabolic genes showed that increased chromatin openness does not mean increased transcription levels.(4)Using CRISPR/d Cas9 to target the human-derived histone acetylase p300 to the gene bre F promoter,we successfully increased the transcription level of bre F gene by 2-3.5-fold and the activation efficiency was correlated with the position of sg RNA.p300-d Cas9 could enhance the transcription of Aspergillus niger FGSC A1279 fumonisin gene fuml and ultimately raise the production of Fumonisin B2 by 12-fold.The effect of epimodifying enzymes of Aspergillus niger’s own origin on bre F gene transcription was also investigated.histone acetylase Gcn E and histone deacetylases Rpd A and Hos A had a repressive effect on bre F transcription,while histone demethylase Lde had no significant effect on the transcription of bre F.hos A-d Cas9 significantly increased the transcription level of the pigment gene fwn A,resulting in an increase in pigment synthesis.