Insights into the Underlying Mechanism of Ochratoxin A Production in Aspergillus niger CBS 513.88 Using Different Carbon Sources

Aspergillus niger produces carcinogenic ochratoxin A (OTA), a serious food safety and human health concern. Here, the ability of A. niger CBS 513.88 to produce OTA using different carbon sources was investigated and the underlying regulatory mechanism was elucidated. The results indicated that 6% sucrose, glucose, and arabinose could trigger OTA biosynthesis and that 1586 differentially expressed genes (DEGs) overlapped compared to a non-inducing nutritional source, peptone. The genes that participated in OTA and its precursor phenylalanine biosynthesis, including pks, p450, nrps, hal, and bzip, were up-regulated, while the genes involved in oxidant detoxification, such as cat and pod, were down-regulated. Correspondingly, the activities of catalase and peroxidase were also decreased. Notably, the novel Gal4-like transcription factor An12g00840 (AnGal4), which is vital in regulating OTA biosynthesis, was identified. Deletion of AnGal4 elevated the OTA yields by 47.65%, 54.60%, and 309.23% using sucrose, glucose, and arabinose as carbon sources, respectively. Additionally, deletion of AnGal4 increased the superoxide anion and H2O2 contents, as well as the sensitivity to H2O2, using the three carbon sources. These results suggest that these three carbon sources repressed AnGal4, leading to the up-regulation of the OTA biosynthetic genes and alteration of cellular redox homeostasis, ultimately triggering OTA biosynthesis in A. niger.


Introduction
Aspergillus and Penicillium frequently contaminate nuts and grapes and produce the "group 2B carcinogen" ochratoxin A (OTA), which is a serious threat to human health and causes huge economic losses worldwide [1,2]. In Aspergillus, OTA is dominantly produced by Aspergillus carbonarius and A. ochraceus, and recent studies reported that several A. niger strains produced OTA [3,4]. A. niger is an extremely well-studied and versatile organism, with widespread applications in the production of organic acids, industrial enzymes, and heterologous proteins. A. niger CBS 513.88 was found to have a putative OTA biosynthetic gene cluster by genome sequencing [5]. Further investigation of the OTA biosynthesis in A. niger CBS 513.88 could, therefore, provide the theoretical basis for the development of effective strategies to control mycotoxin contamination in the fermentation of industrial products.

Identification of the OTA-Producing Aptitude by A. niger CBS 513.88
To investigate whether the A. niger CBS 513.88 strain could produce OTA, the retention time of the OTA standard was determined by HPLC-FLD ( Figure 1A). The corresponding peak was observed at a similar time in the methanolic extracts of A. niger CBS 513.88 that was cultured on YES solid media under the same chromatographic conditions ( Figure 1B), so the peak was further analyzed using UHPLC-MS. The results confirmed that A. niger CBS 513.88 could produce OTA ( Figure 1C,D).

Influence of Different Carbon Sources on Colony Morphology, Conidia Production, and OTA Biosynthesis of A. niger CBS 513.88
After 5.5 days of culture, colony morphology and sporulation ability were significantly changed in A. niger CBS 513.88 when grown on yeast extract containing different carbon sources (sucrose, glucose, and arabinose), when compared with peptone. As shown in Figure 2A, on YEP solid medium, colonies were covered by dense dark brown conidial heads in the center. In contrast, on YES, YEG, and YEA solid media, colonies were light yellow. The conidia productions on YES, YEG, and YEA solid media were significantly lower than that on YEP solid medium ( Figure 2B). Moreover, the colony diameters on YES, YEG, and YEA solid media were larger than that on YEP solid medium ( Figure 2C). Together, the results indicated that the growth rates of the strains grown on YES, YEG, and YEA solid media were higher than that on YEP solid medium.

Influence of Different Carbon Sources on Colony Morphology, Conidia Production, a OTA Biosynthesis of A. niger CBS 513.88
After 5.5 days of culture, colony morphology and sporulation ability were cantly changed in A. niger CBS 513.88 when grown on yeast extract containing d carbon sources (sucrose, glucose, and arabinose), when compared with pepto shown in Figure 2A, on YEP solid medium, colonies were covered by dense dark  ). Together, the results indicated that the growth rates of the strains grown on YES, YEG, and YEA solid media were higher than that on YEP solid medium. The influence of different carbon sources on OTA biosynthesis was further studied by HPLC-FLD. After 5.5 days of cultivation at 23 °C in the dark, the concentration of OTA reached around 4.19, 3.64, and 21.93 µg/g dry hyphae on YES, YEG, and YEA solid media, respectively. However, OTA was not detected on the YEP solid medium ( Figure 2D). These results confirmed that arabinose was the best carbon source to produce OTA in A. niger CBS 513.88, followed by sucrose and glucose, while peptone inhibited OTA production. Arabinose, sucrose, and glucose were, therefore, defined as OTA-inducing carbon sources, and peptone as the non-inducing nutritional source.

Transcriptional Profiles and DEGs Analyses of A. niger CBS 513.88 Using Different Carbon Sources
To further reveal the mechanism of OTA production using different carbon sources, we performed transcriptome sequencing of the A. niger strain. Strains grown on YES, YEG, and YEA solid media were the test group, and YEP medium was the control group. We identified DEGs, which overlapped with the three OTA-inducing carbon sources and the non-inducing nutritional source, to narrow the scope of our studies. The samples were all analyzed at 5.5 days of incubation at 23 °C in the dark. Transcriptome analyses showed that 1778, 1618, and 1792 genes were significantly up-regulated on YES, YEG, and YEA solid media, respectively, whereas 1317, 1079, and 1579 genes were significantly downregulated, respectively ( Figure 3A). A total of 1586 DEGs overlapped on YES, YEG, and YEA solid media, of which 986 were up-regulated and 595 were down-regulated ( Figure  3B). Among them, genes associated with the OTA precursor phenylalanine (An08g06800, The influence of different carbon sources on OTA biosynthesis was further studied by HPLC-FLD. After 5.5 days of cultivation at 23 • C in the dark, the concentration of OTA reached around 4.19, 3.64, and 21.93 µg/g dry hyphae on YES, YEG, and YEA solid media, respectively. However, OTA was not detected on the YEP solid medium ( Figure 2D). These results confirmed that arabinose was the best carbon source to produce OTA in A. niger CBS 513.88, followed by sucrose and glucose, while peptone inhibited OTA production. Arabinose, sucrose, and glucose were, therefore, defined as OTA-inducing carbon sources, and peptone as the non-inducing nutritional source.

Transcriptional Profiles and DEGs Analyses of A. niger CBS 513.88 Using Different Carbon Sources
To further reveal the mechanism of OTA production using different carbon sources, we performed transcriptome sequencing of the A. niger strain. Strains grown on YES, YEG, and YEA solid media were the test group, and YEP medium was the control group. We identified DEGs, which overlapped with the three OTA-inducing carbon sources and the non-inducing nutritional source, to narrow the scope of our studies. The samples were all analyzed at 5.5 days of incubation at 23 • C in the dark. Transcriptome analyses showed that 1778, 1618, and 1792 genes were significantly up-regulated on YES, YEG, and YEA solid media, respectively, whereas 1317, 1079, and 1579 genes were significantly down-regulated, respectively ( Figure 3A). A total of 1586 DEGs overlapped on YES, YEG, and YEA solid media, of which 986 were up-regulated and 595 were down-regulated ( Figure 3B). Among them, genes associated with the OTA precursor phenylalanine (An08g06800, An14g06010, and An15g02460) and OTA biosynthesis (nrps, P450, hal, and bzip) were significantly upregulated (Table 1), which was confirmed by qRT-PCR analyses ( Figure S1). Although the transcriptional level of pks was unchanged on YES and YEG media, it was significantly up-regulated on YEA medium with the highest OTA concentration. Together, the results showed that carbon sources affected the OTA production of A. niger by controlling the levels of OTA and its precursor phenylalanine biosynthesis. An14g06010, and An15g02460) and OTA biosynthesis (nrps, P450, hal, and bzip) were significantly up-regulated (Table 1), which was confirmed by qRT-PCR analyses ( Figure S1). Although the transcriptional level of pks was unchanged on YES and YEG media, it was significantly up-regulated on YEA medium with the highest OTA concentration. Together, the results showed that carbon sources affected the OTA production of A. niger by controlling the levels of OTA and its precursor phenylalanine biosynthesis.   Catalase activity (cat) −2.65 *** −2.04 *** −3.21 *** An09g00820 Predicted peroxidase activity (pod) −2.04 *** −2.40 *** −2.75 *** An16g00630 Predicted peroxidase activity (pod) −2.16 ** −3.67 *** −1.59 * All data are the mean ± standard deviation of independent tests performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001.
KEGG pathway and GO analyses for the 1586 DEGs were also conducted. According to the KEGG pathway analyses, DEGs were involved in carbohydrate metabolism, containing glycolysis/gluconeogenesis, and starch/sucrose/galactose/amino acids metabolism ( Figure 3C). Genes involved in phenylalanine, histidine, tyrosine, tryptophan, and arginine biosynthesis were up-regulated (Table 1). According to GO terms analyses, DEGs were primarily involved in oxidative stress. In the molecular functions and biological processes category, DEGs were mainly associated with oxidoreductase activity and oxidation-reduction processes, respectively ( Figure 3D).

Carbon Sources Affect the Cellular Redox Homeostasis of A. niger CBS 513.88
The carbon source affects the cellular redox status in filamentous fungi, and OTA production is closely related to oxidative stress [3,10,12]. The generated H 2 O 2 can be catalyzed by CAT and POD. In this study, the expression levels of An08g08920 and An14g00690 that encode CAT and An09g00820 and An16g00630 that encode POD, were all significantly down-regulated in three OTA-inducing conditions, when compared to non-inducing conditions ( Figure 4A) (Table 1). Correspondingly, the activities of the two antioxidant enzymes of A. niger strains were detected ( Figure 4B). Compared to that on YEP solid medium, the activities of CAT on YES, YEG, and YEA solid medium decreased by 75.6%, 69.5%, and 62.8%, respectively. Similarly, compared to that on the YEP solid medium, the activities of POD on YES, YEG, and YEA solid medium decreased by 40.4%, 48.0%, and 64.6%, respectively. Together, the results indicated that carbon sources may affect OTA biosynthesis by altering the homeostasis of cellular redox. medium, the activities of POD on YES, YEG, and YEA solid medium decreased by 40.4%, 48.0%, and 64.6%, respectively. Together, the results indicated that carbon sources may affect OTA biosynthesis by altering the homeostasis of cellular redox. All data are expressed as the mean ± standard deviation of independent tests performed in triplicate (*** t < 0.001).

Disruption of a Novel Gal4-like Transcription Factor Affects OTA Biosynthesis
Transcription factors (TFs) in gene regulatory networks have previously been reported to regulate intracellular metabolic processes in response to environmental changes [23]. There were 25 up-regulated TFs and 14 down-regulated TFs in the 3 OTA-inducing conditions (Table S1). Among them, 20 TFs had homologous genes in yeast and were annotated according to the Saccharomyces Genome Database, SGD (https://www.yeastgenome.org/, accessed on 1 December 2021). Using SGD annotation, these TF genes were shown to be involved in carbon metabolism, stress response, amino acid catabolism, sterol biosynthesis, etc. An12g00840, a fungal-specific Gal4-like Zn(II)2Cys6 transcription factor (AnGal4), was significantly down-regulated in three OTA-inducing carbon sources. To characterize the role of AnGal4 in OTA biosynthesis, an AnGal4 deletion strain was constructed ( Figure 5A). The samples were collected after 5.5 days at 23 °C, when grown in the three OTA-inducing conditions. The results showed OTA yields of the ΔAnGal4 strain All data are expressed as the mean ± standard deviation of independent tests performed in triplicate (*** t < 0.001).

Disruption of a Novel Gal4-like Transcription Factor Affects OTA Biosynthesis
Transcription factors (TFs) in gene regulatory networks have previously been reported to regulate intracellular metabolic processes in response to environmental changes [23]. There were 25 up-regulated TFs and 14 down-regulated TFs in the 3 OTA-inducing conditions (Table S1). Among them, 20 TFs had homologous genes in yeast and were annotated according to the Saccharomyces Genome Database, SGD (https://www.yeastgenome.org/, accessed on 1 December 2021). Using SGD annotation, these TF genes were shown to be involved in carbon metabolism, stress response, amino acid catabolism, sterol biosynthesis, etc. An12g00840, a fungal-specific Gal4-like Zn(II)2Cys6 transcription factor (AnGal4), was significantly down-regulated in three OTA-inducing carbon sources. To characterize the role of AnGal4 in OTA biosynthesis, an AnGal4 deletion strain was constructed ( Figure 5A). The samples were collected after 5.5 days at 23 • C, when grown in the three OTA-inducing conditions. The results showed OTA yields of the ∆AnGal4 strain increased by 47.65%, 54.60%, and 309.23% on YES, YEG, and YEA solid media, respectively ( Figure 5B). Correspondingly, the transcript levels of OTA biosynthetic genes of the ∆AnGal4 strain were all

AnGal4 Affects Cellular Redox Homeostasis of A. niger
To further investigate the correlation between AnGal4 and oxidative stress, the O 2− and H2O2 contents, and the sensitivity to H2O2 in the ΔAnGal4 strain using three carbon sources were determined. The results showed that the O 2− contents in the ΔAnGal4 strain increased by 158.37%, 80.57%, and 27.07% on YES, YEG, and YEA solid media, respectively ( Figure 6A). The H2O2 contents in ΔAnGal4 strain increased by 11.94%, 25.77%, and 20.41% on YES, YEG, and YEA solid media, respectively ( Figure 6B). In addition, although the growth was unchanged in the ΔAnGal4 and control strains under unstressed conditions, the ΔAnGal4 strain exhibited significant growth defects when supplemented with 10, 20 and 30 mM H2O2, with the defects especially pronounced at high concentrations, showing that deletion of ΔAnGal4 increased the sensitivity to H2O2 ( Figure 6C). Together, the results suggested that AnGal4 affected the cellular redox homeostasis of A. niger and that the deletion of ΔAnGal4 increased cellular oxidative stress.

AnGal4 Affects Cellular Redox Homeostasis of A. niger
To further investigate the correlation between AnGal4 and oxidative stress, the O 2− and H 2 O 2 contents, and the sensitivity to H 2 O 2 in the ∆AnGal4 strain using three carbon sources were determined. The results showed that the O 2− contents in the ∆AnGal4 strain increased by 158.37%, 80.57%, and 27.07% on YES, YEG, and YEA solid media, respectively ( Figure 6A). The H 2 O 2 contents in ∆AnGal4 strain increased by 11.94%, 25.77%, and 20.41% on YES, YEG, and YEA solid media, respectively ( Figure 6B). In addition, although the growth was unchanged in the ∆AnGal4 and control strains under unstressed conditions, the ∆AnGal4 strain exhibited significant growth defects when supplemented with 10, 20 and 30 mM H 2 O 2 , with the defects especially pronounced at high concentrations, showing that deletion of ∆AnGal4 increased the sensitivity to H 2 O 2 ( Figure 6C). Together, the results suggested that AnGal4 affected the cellular redox homeostasis of A. niger and that the deletion of ∆AnGal4 increased cellular oxidative stress.

Discussion
A. niger CBS 513.88 is a "generally recognized as safe" industrial fermentation strain, and previous reports showed the presence of potential OTA biosynthesis genes in its genome [5]. We found that CBS 513.88 produced OTA, and confirmed that arabinose was the suitable carbon source for OTA production, followed by glucose and sucrose. Our results are consistent with those of Hashem et al. [16], who reported that glucose and sucrose were favorable carbon sources for OTA production, but inconsistent with those of Wang et al. [24], who reported that arabinose repressed OTA production in A. ochraceus. The differences might be attributable to the different physiological and genetic characteristics of the fungi [25]. In addition, we found that peptone suppressed OTA production.

Discussion
A. niger CBS 513.88 is a "generally recognized as safe" industrial fermentation strain, and previous reports showed the presence of potential OTA biosynthesis genes in its genome [5]. We found that CBS 513.88 produced OTA, and confirmed that arabinose was the suitable carbon source for OTA production, followed by glucose and sucrose. Our results are consistent with those of Hashem et al. [16], who reported that glucose and sucrose were favorable carbon sources for OTA production, but inconsistent with those of Wang et al. [24], who reported that arabinose repressed OTA production in A. ochraceus. The differences might be attributable to the different physiological and genetic characteristics of the fungi [25]. In addition, we found that peptone suppressed OTA production. The inhibitory effect of peptone on mycotoxins may be attributed to the limited availability of malonyl-CoA or acetyl, because almost all of them were used for primary energy production rather than mycotoxin biosynthesis [26]. In A. flavus, YES medium induced aflatoxin biosynthesis, while YEP medium repressed it, consistent with OTA production in A. niger. Sequencing of the transcriptomes of the A. niger CBS 513.88 strain cultured in OTA-inducing conditions (YEA, YES, YEG) compared to non-inducing conditions (YEP) was performed. The common differences were investigated to elucidate the underlying regulatory mechanism.
The OTA biosynthetic pathway has been well characterized in A. ochraceus [3]. Four genes, nrps, p450, hal, and bzip in the OTA biosynthetic gene cluster, were all up-regulated when sucrose or glucose was used as the sole carbon source. More importantly, pks, the first and backbone gene in the OTA biosynthesis pathway, was significantly up-regulated with arabinose as the carbon source. The high expression of pks was an important reason for the highest OTA yield when arabinose was the carbon source in A. niger. The results indicated that carbon sources affected OTA biosynthesis by regulating the expression of OTA biosynthesis-associated genes in A. niger.
Medina et al. [25] and Abbas et al. [27] reported that amino acids, including phenylalanine, tyrosine, tryptophan, histidine, and arginine, induced OTA production. Phenylalanine, as the precursor for OTA biosynthesis, is sequentially synthesized via the shikimate and phenylalanine biosynthesis pathways [25]. Our results showed that An08g06800 (catalyzing steps two to six in the shikimate pathway), An14g06010 (catalyzing step eight of the phenylalanine biosynthesis), and An15g02460 (catalyzing step 10 of the phenylalanine biosynthesis), were all up-regulated in the presence of three OTA-inducing carbon sources. Additionally, the genes involved in tyrosine, tryptophan, histidine, and arginine biosynthesis were also up-regulated in the presence of three OTA-inducing carbon sources. Together, the results showed that carbon sources may affect OTA biosynthesis by regulating the biosynthesis of amino acids.
Oxidative stress is an important factor correlated with the biosynthesis of mycotoxins in fungi [1,[28][29][30][31]. Reverberi et al. [32] suggested that oxidative stress agents, such as carbon tetrachloride or t-butyl hydroperoxide, induced OTA production in A. ochraceus, while antioxidants had the opposite effects [33]. In addition, oxidative stress can regulate OTA biosynthesis, which has been confirmed by the disruption of the oxidative stressrelated genes, Aoyap1 and AoloxA [33]. Our results showed that expression of the oxidant detoxification-related genes (cat and pod) and activities of the corresponding antioxidant enzymes (CAT and POD) on the three OTA-inducing carbon sources were lower overall than that on the non-inducing nutritional source. These results suggested that carbon sources may trigger OTA biosynthesis by altering cell redox homeostasis in A. niger, consistent with the observed association of mannose blocking fumonisin production with milder oxidative stress in Fusarium proliferatum [12].
A carbon source is a principal factor that regulates various metabolic processes via TFs [6,23]. We identified 39 significantly changed TFs that overlapped in the 3 OTAinducing conditions (YEA, YES, YEG) compared to non-inducing conditions (YEP). These mainly included ZnCys, C 2 H 2 , NEG and the bZIP family (Table S1). Among them, BrlA, a transcriptional activator for conidiation [34], was significantly down-regulated in the presence of the three OTA-inducing carbon sources. The findings indicate that the conidiation was inhibited. In addition, Zn(II)2Cys6 proteins are always involved in the biosynthesis of fungal secondary metabolites. For example, Zn(II)2Cys6 proteins AflR, Fum21, GliZ, MclR, CtnA, and Gip2 are transcriptional activators for the production of the fungal secondary metabolites aflatoxin [35], fumonisin [36], gliotoxin [37], compactin [38], citrinin [39], aurofusarin [40], respectively. Our transcriptome analysis indicated that An12g00840, a fungal specific transcription factor with a Gal4-like Zn(II)2Cys6 binuclear cluster DNA-binding domain (AnGal4), was significantly down-regulated in the presence of three OTA-inducing carbon sources. Based on the above findings, we speculate that the AnGal4 TF may be involved in the biosynthesis of OTA. Our results indicate that disruption of AnGal4 significantly increased OTA yield, and the expressions of pks, nrps, p450, bzip, and hal, in three OTA-inducing carbon sources. Specifically, the OTA yields of the ∆AnGal4 strain increased by 47.65%, 54.60%, and 309.23%, when sucrose, glucose, and arabinose were used as the sole carbon source, respectively. The results indicate that AnGal4 activity mediated by the carbon source negatively regulated the OTA biosynthesis. There is only very limited information, including carbon catabolite repression (CCR) [17], cAMP signaling pathway [18], and the flux rearrangement of primary metabolism [19], concerning the involvement in the regulation of secondary metabolite production responding to different carbon sources. Among them, CCR genes CreA and Cre1 regulate the production of several secondary metabolites, including cephalosporin C [41], lovastatin [42], aflatoxin [43], and OTA [24]. In our study, the novel TF AnGal4 was identified for the first time and demonstrated to have a negative regulatory role in OTA biosynthesis responding to the different carbon sources in A. niger. In addition, a previous report suggested that Gal4 of yeast bound to upstream activation sites of target genes with 5 -CGGN 5 (T/A)N 5 CCG-3 in vivo, or (A/C)GGN 10-12 CCG in vitro [43]. The promoters of the OTA biosynthesis genes of A. niger were predicted using a dedicated promoter prediction website (https://services.healthtech.dtu.dk, accessed on 1 February 2022). Notably, we found that AnGal4 had direct binding sites on the promoters of the OTA biosynthesis genes pks, nrps, p450, bzip, and hal, in vivo or in vitro (Table S3).
The results indicate that AnGal4 may directly regulate OTA biosynthesis by binding to the OTA biosynthesis genes.
Previous reports indicated that the CCR gene CreA homologue gene Mig1 is negatively correlated with the oxidative stress response [44]. We speculate that AnGal4 regulated by the carbon source may be involved in the oxidative stress. Our results showed that deletion of AnGal4 increased the contents of O 2 − and H 2 O 2 , as well as the sensitivity to H 2 O 2 in the presence of the three selected carbon sources, demonstrating that AnGal4 alters the cellular redox homeostasis in A. niger. The ∆AnGal4 strain had the highest O 2 − and H 2 O 2 concentrations in YEA medium, consistent with the highest OTA yield. The results suggest that carbon source-mediated AnGal4 may regulate OTA biosynthesis by altering cellular redox homeostasis.

Conclusions
Our results confirm that arabinose, sucrose, and glucose contribute to OTA production in the industrial fermentation strain A. niger CBS 513.88, and characterize the novel Gal4-like TF An12g00840 (AnGal4). The results suggest that carbon sources repress AnGal4, leading to up-regulation of OTA biosynthetic genes and altered cellular redox homeostasis. These changes ultimately trigger OTA biosynthesis in A. niger CBS 513.88. This study clarifies the underlying regulatory mechanism of OTA biosynthesis responding to different carbon sources. The findings provide a theoretical basis for the development of effective strategies to control mycotoxin contamination in the fermentation of industrial products.

Construction of Strains and Cultivation Conditions
The strains and primers used in this study are listed in Table 2 and Supplementary  Table S2, respectively. A. niger CBS 513.88 (provided by professor Li Pan, South China University of Technology) and A. niger CBS 513.88 (kusA − , pyrG − ) (laboratory constructed) were grown on YEA (6% arabinose, 2% yeast extract, pH 5.8), YES (6% sucrose, 2% yeast extract, pH 5.8), YEG (6% glucose, 2% yeast extract, pH 5.8), and YEP (6% peptone, 2% yeast extract, pH 5.8) solid media that were not supplemented or supplemented with 10 mM uridine. In addition, the YEA, YES, YEG, and YEP solid media were also used for strain morphology and conidia assay, OTA production, enzyme activity assay, determinations of superoxide anion and hydrogen peroxide (H 2 O 2 ) contents, and the H 2 O 2 susceptibility assays. Czapek-Dox (CD) medium supplemented with or without uridine was used for strain transformation. All strains were cultivated at 30 • C for 5.5 days for growth and at 23 • C for 5.5 days in the dark for OTA production. AnGal4 (An12g00840) gene knockout was performed by homologous recombination [45], and A. niger CBS 513.88 (kusA − , pyrG + ) was the control strain. The pyrG gene expression cassette was used as the selection marker, which was cloned from plasmid ANIp7. Homologous fragments of 1.60 kb and 1.47 kb at either end of the AnGal4 open reading frame (ORF) were amplified. The upstream and downstream homologous fragments and pyrG expression cassette were ligated into plasmid pMD20 using a one-step cloning kit (Novazyme, Nanjing, China) to yield pMD20-∆AnGal4, which was transferred into the A. niger CBS 513.88 (kusA − , pyrG − ) strain. The recombinant strain was screened using Czapek-Dox (CD) medium lacking uridine. The screening was verified by PCR using the genome of the correct transformants as a template.

Colony Morphology, Mycelial Growth, and Conidia Production
Conidia suspensions (5 µL, 10 6 spores/mL) were centrally spotted on YEA, YES, YEG, and YEP media and cultivated for 5.5 days at 23 • C. Colony morphology and colony diameters were observed and measured [46]. Conidia suspensions were viewed using a microscope and counted using a hemocytometer. All cultures were conducted in triplicate.

High-Performance Liquid Chromatography (HPLC) Analysis of OTA
Conidia suspensions (200 µL, 10 6 spores/mL) were spread evenly on YEA, YES, YEG, and YEP media and cultivated for 5.5 days at 23 • C in the dark. All mycelium and agar on plates were collected, and then were mashed and placed in an Erlenmeyer flask with 25 mL methanol, followed by shaking for 1 h in the dark. All cultures were conducted in triplicate. OTA was detected by HPLC with fluorescence detection (HPLC-FLD) (Agilent Technologies, San Jose, CA, USA) [47].

Ultra-High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS) Analysis
For UHPLC-MS analysis, the supernatant of the OTA extract was purified using an OTA immunoaffinity column (PriboFast, Beijing, China) and transferred to a 2 mL Eppendorf tube. The highly purified sample was detected using a Q-Orbitrap instrument system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a binary solvent delivery system. A C18 column (2.1 × 100 mm, 1.7 µm particle diameter; Thermo Fisher Scientific) was used for chromatography. The UHPLC-MS run used positive ESI. The details were performed as previously described [48].

Transcriptome Analysis
Conidia suspensions (200 µL, 10 6 spores/mL) were spread evenly on YEA, YES, YEG, and YEG media and cultivated for 5.5 days at 23 • C in the dark. Hyphae were collected, then frozen in liquid nitrogen and subjected to transcriptome analyses. We first extracted total RNA using a UNIQ-10 TRIzol RNA Purification Kit (Sangon Biotech, Shanghai, China), with three biological parallels for each sample, then sequenced it using a NovaSeq (Personalbio, Nanjing, China). Raw data showed significant differences between YES and YEP, YEG and YEP, and YEA and YEP, with a bioproject accession number of PRJNA765465. Significant differences were indicated by an absolute log 2 -fold change of 1.0 or greater, and p-values of 0.05 or less. All annotations were obtained according to NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 10 December 2021). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed according to the reference [49], and were classified as significantly enriched among the differentially expressed genes (DEGs).

Real-Time Quantitative PCR (RT-qPCR) Analysis
The qPCR data were analyzed according to a previously described method [50]. After RNA extraction, we used a PrimeScript™ RT reagent Kit (TaKaRa Bio, Beijing, China) to obtain the cDNA. We then used qgpdA as the reference gene, and the equation N = 2 −∆∆Ct to evaluate the relative transcription levels. The t-test was performed to assess the differences between samples.

Determination of Antioxidant Enzyme Activity, and Superoxide Anion and H 2 O 2 Contents
Two hundred microliter aliquots of conidia suspensions containing 10 6 spores/mL were spread evenly on YEA, YES, YEG, and YEP media and cultivated for 5.5 days at 23 • C in the dark. The mycelia were collected, triturated with acetone, and the supernatant was collected by centrifugation at 4 • C. Catalase (CAT) and glutathione peroxidase (POD) were quantified using the corresponding detection kits (Solarbio, Beijing, China). The superoxide anion (O 2− ) and H 2 O 2 contents were also measured according to the corresponding detection kit instructions (Solarbio, Beijing, China).

Assay of H 2 O 2 Susceptibility
Five microliter aliquots of conidia suspensions containing 10 6 spores/mL were spotted onto YEA, YES, YEG, and YEP media supplemented with H 2 O 2 at different concentrations (0, 10, 20, and 30 mM), and cultivated for 5.5 days at 30 • C. The growth of strains on plates was then recorded.

Statistical Analysis
All experiments were repeated with three independent biological replicates. The data are expressed as the mean ± standard deviation of independent tests performed in triplicate. The differences were calculated with student's t test (t-test), and marked with *, **, and *** as t < 0.05, t < 0.01, and t < 0.001, respectively.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/toxins14080551/s1, Figure S1: qRT-PCR analyses of differentially expressed genes in OTA precursor phenylalanine and OTA biosynthesis; Table S1: The significantly changed TFs that overlapped in the three OTA-inducing conditions (YEA, YES, YEG) compared to non-inducing condition (YEP); Table S2: All primers used in this study; Table S3: Sequences of Gal4 binding sites on the predicted promoter.

Conflicts of Interest:
The authors declare no conflict of interest.