The Regulation Mechanism Of Ochratoxin A Biosynthesis In Aspergillus Niger CBS 513.88 Using Carbon Sources

Aspergillus niger is widely used in food industry.However,recent researches demonstrate that several A.niger strains could produce strong carcinogenic toxins such as ochratoxin A（OTA）,posing a serious threat to food safety and human health.OTA biosynthesis is affected by many factors,including carbon sources,temperature,pH,oxidative stress and metal ions,among which carbon sources is one of the most important factors affecting OTA biosynthesis.At present,the impact of carbon sources on OTA biosynthesis has been reported,however,the biosynthetic pathways and regulatory mechanisms of carbon sources affecting OTA is far from being elucidated.Especially in A.niger,there are few studies on the biosynthetic pathway and regulatory mechanism of OTA.Therefore,it is urgent to further explore the OTA biosynthesis pathway in A.niger and the regulatory mechanism of OTA by carbon sources.It provides a theoretical basis for the prevention,control,early warning and detoxification of OTA pollution.It is of great significance to ensure the safety of A.niger strains in industrial production and protect the health of humans and animals.In this study,A.niger CBS 513.88 was taken as the research object,the OTA biosynthetic regulatory network was the center,and the screening of OTA-induced carbon sources was used as the starting point,and the function of differentially expressed genes under different carbon source conditions was analyzed through biological research methods such as transcriptome sequencing and gene knockout,and the regulatory mechanism of carbon sources on OTA biosynthesis in A.niger was comprehensively analyzed from multiple perspectives,such as morphology,physiology and genetics.The main findings are as follows:（1）A.niger CBS 513.88 strains were identified as OTA-producing fungi by HPLC-FLD and UHPLC-MS,and their OTA biosynthesis ability under different carbon source culture conditions was studied.The results showed that 6%sucrose,glucose and arabinose could induce OTA biosynthesis,and peptone was an inducible carbon source to inhibit OTA biosynthesis.（2）The transcriptome sequencing results of A.niger CBS 513.88 under four carbon source culture conditions showed that compared with peptone,the three carbon sources had1586 common differential expression genes.Among them,genes involved in the biosynthesis of OTA and its precursor phenylalanine,including pks,p450,nrps,hal and the bzip gene for a transcription factor,were significantly upregulated,while genes involved in oxidative detoxification,such as cat and pod,were significantly downregulated.QRT-PCR validation and activity determination of catalase（CAT）and peroxidase（POD）were consistent with transcriptomic results.In transcriptome data analysis,it was found that transcription factors such as An12g00840（AnGal4）and An15g00120（AnAzf1）were significantly expressed compared with controls.（3）The role of AnGal4 in the OTA biosynthetic pathway was analyzed.The results showed that the deletion of AnGal4 increased the biosynthesis of OTA in A.niger strains when sucrose,glucose and arabinose were used as carbon sources.In addition,under these three carbon source culture conditions,the deletion of AnGal4 increased the content of O₂.^-and H₂O₂,as well as increased the sensitivity to H₂O₂.These results suggested that these three carbon sources inhibited the expression of AnGal4,led to the upregulation of OTA biosynthesis-related genes and the change of cellular redox homeostasis,and ultimately induced the biosynthesis of OTA in A.niger.（4）The AnAzf1 gene in A.niger was knocked out by homologous recombination and the effect of its deletion strain on OTA biosynthesis was studied.Compared with the control group,AnAzf1 deletion completely blocked the biosynthesis of OTA in A.niger and inhibited the expression of key genes p450,nrps,hal and bzip in the OTA biosynthetic gene cluster at the transcription level.The results indicated that AnAzf1 was a positive regulator of OTA biosynthesis.（5）The regulation mechanism by which AnAzf1 deficiency inhibits OTA biosynthesis in A.niger strains.The transcriptome sequencing results of AnAzf1-deficient strain and control strain of A.niger showed that AnAzf1 deletion led to upregulation of antioxidant-related genes,downregulation of oxidative phosphorylation-related genes,increased activity of CAT and POD related to the removal of reactive oxygen species（ROS）,and decreased corresponding ROS levels,indicating that AnAzf1 deletion affected cellular redox homeostasis.The upregulation of genes（cat,catA,hog1 and gfd）in the MAPK pathway and downregulation of iron homeostasis-related genes significantly reduced the activity and ATP content of enzymes involved in mitochondrial energy metabolism,including complex I（NADH-ubiquinone oxidoreductase）,complex V（ATP synthase）and NAD-malate dehydrogenase（NAD-MDH）,indicating that AnAzf1 deletion affected cellular oxidative phosphorylation levels,MAPK pathway and cellular iron homeostasis.Overall,AnAzf1 affected the biosynthesis of OTA in A.niger by regulating cellular ROS accumulation and oxidative phosphorylation levels.In addition,the deletion of AnAzf1 led to increased sensitivity of A.niger to essential oils,suggesting that AnAzf may be a target for the bacteriostatic effects of plant essential oils.AnAzf1 was further confirmed in a medium based on maize and peanut to positively regulate the biosynthesis of OTA in A.niger.In summary,this study reveals the regulatory mechanism of carbon source influence on OTA synthesis in A.niger CBS 513.88,which provides a theoretical basis for the prevention and control of OTA pollution.