Fus3 Interacts with Gal83, Revealing the MAPK Crosstalk to Snf1/AMPK to Regulate Secondary Metabolic Substrates in Aspergillus flavus.

Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi such as Aspergillus flavus. Essential for the fungi to respond to environmental changes and aflatoxin synthesis, the pheromone mitogen-activated protein kinase (MAPK) is a potential regulator of aflatoxin biosynthesis. However, the mechanism by which pheromone MAPK regulates aflatoxin biosynthesis is not clear. Here, we showed Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway as a regulator of the Snf1/AMPK energy-sensing pathway modulating aflatoxins synthesis substrates. The screening for Fus3 target proteins identified the ß subunit of Snf1/AMPK complexes using tandem affinity purification and multiomics. This subunit physically interacted with Fus3 both in vivo and in vitro and received phosphorylation from Fus3. Although the transcript levels of aflatoxin synthesis genes were not noticeably downregulated in both gal83 and fus3 deletion mutant strains, the levels of aflatoxin B1 and its synthesis substrates and gene expression levels of primary metabolizing enzymes were significantly reduced. This suggests that both the Fus3-MAPK and Snf1/AMPK pathways respond to energy signals. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking with the Snf1/AMPK complexes.

Antifungal potential of lipopeptides produced by the Bacillus siamensis Sh420 strain against Fusarium graminearum.

Biological control is a more sustainable and environmentally friendly alternative to chemical fungicides for controlling Fusarium spp. infestations. In this work, Bacillus siamensis Sh420 isolated from wheat rhizosphere showed a high antifungal activity against Fusarium graminearum as a secure substitute for fungicides. Sh420 was identified as B. siamensis using phenotypic evaluation and 16S rDNA gene sequence analysis. An in vitro antagonistic study showed that Sh420's lipopeptide (LP) extract exhibited strong antifungal properties and effectively combated F. graminearum. Meanwhile, lipopeptides have the ability to decrease ergosterol content, which has an impact on the overall structure and stability of the plasma membrane. The PCR-based screening revealed the presence of antifungal LP biosynthetic genes in this strain's genomic DNA. In the crude LP extract of Sh420, we were able to discover several LPs such as bacillomycin, iturins, fengycin, and surfactins using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Microscopic investigations (fluorescent/transmission electron microscopy) revealed deformities and alterations in the morphology of the phytopathogen upon interaction with LPs. Sh420 LPs have been shown in grape tests to be effective against F. graminearum infection and to stimulate antioxidant activity in fruits by avoiding rust and gray lesions. The overall findings of this study highlight the potential of Sh420 lipopeptides as an effective biological control agent against F. graminearum infestations.IMPORTANCEThis study addresses the potential of lipopeptide (LP) extracts obtained from the strain identified as Bacillus siamensis Sh420. This Sh420 isolate acts as a crucial player in providing a sustainable and environmentally friendly alternative to chemical fungicides for suppressing Fusarium graminearum phytopathogen. Moreover, these LPs can reduce ergosterol content in the phytopathogen influencing the overall structure and stability of its plasma membrane. PCR screening provided confirmation regarding the existence of genes responsible for biosynthesizing antifungal LPs in the genomic DNA of Sh420. Several antibiotic lipopeptide compounds were identified from this bacterial crude extract using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Microscopic investigations revealed deformities and alterations in the morphology of F. graminearum upon interaction with LPs. Furthermore, studies on fruit demonstrated the efficacy of Sh420 LPs in mitigating F. graminearum infection and stimulating antioxidant activity in fruits, preventing rust and gray lesions.

AwSclB regulates a network for Aspergillus westerdijkiae asexual sporulation and secondary metabolism independent of the fungal light control.

As a prevalent pathogenic fungus, Aspergillus westerdijkiae poses a threat to both food safety and human health. The fungal growth, conidia production and ochratoxin A (OTA) in A. weterdijkiae are regulated by many factors especially transcription factors. In this study, a transcription factor AwSclB in A. westerdijkiae was identified and its function in asexual sporulation and OTA biosynthesis was investigated. In addition, the effect of light control on AwSclB regulation was also tested. The deletion of AwSclB gene could reduce conidia production by down-regulation of conidia genes and increase OTA biosynthesis by up-regulation of cluster genes, regardless under light or dark conditions. It is worth to note that the inhibitory effect of light on OTA biosynthesis was reversed by the knockout of AwSclB gene. The yeast one-hybrid assay indicated that AwSclB could interact with the promoters of BrlA, ConJ and OtaR1 genes. This result suggests that AwSclB in A. westerdijkiae can directly regulate asexual conidia formation by activating the central developmental pathway BrlA-AbaA-WetA through up-regulating the expression of AwBrlA, and promote the light response of the strain by activating ConJ. However, AwSclB itself is unable to respond to light regulation. This finding will deepen our understanding of the molecular regulation of A. westerdijkiae development and secondary metabolism, and provide potential targets for the development of new fungicides.

Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield.

Fungi and subsequent mycotoxins contamination in agricultural products have caused enormous losses and great harm to human and animal health. Biological control has attracted the attention of researchers due to its advantages, including mild conditions, low cost, high efficiency and low nutrient loss. In this study, a newly isolated strain Bacillus amyloliquefaciens A-1 (A-1), was screened for its ability to inhibit the growth and Aflatoxin B1 (AFB1) production of Aspergillus flavus NRRL 3357. Electron microscopy results revealed that mycelium and conidia of A. flavus were destroyed by A-1, affecting hyphae, cell walls, cell membranes and organelles. RNA-seq analysis indicated disturbance in gene expression profiles of A. flavus, including amino acid degradation and starch and sucrose metabolism pathways. Importantly, the biosynthesis of AFB1 was significantly inhibited by the down-regulation of key regulatory genes, aflR and aflS, and the simultaneous down-regulation of most structural genes. Genome analysis predicted six secondary metabolites biosynthetic gene clusters. Then, four surfactin synthesized by cluster C were identified as the main active substance of A-1 using HPLC-Q-TOF-MS. The addition of alanine, threonine, Fe2+ increased surfactin production. Notably, the overexpression of comX also improved surfactin production. The vivo test results indicated that A-1 could significantly inhibit the decay of pear by Aspergillus westerdijkiae, and the mildew of maize and peanuts. Especially, the overexpression of comX in A-1 could enhance the inhibitory activity. In conclusion, the inhibition mechanism of A-1 was revealed, and comX was found can improve the production of surfactin and subsequent activities, which provides the scientific basis for the development of biocontrol agents to reduce spoilage in agricultural products.

Identification of a Novel Bacillus velezensis IS-6 Nudix Hydrolase Nh-9 Involved in Ochratoxin A Detoxification by Transcriptomic Profiling and Functional Verification.

Contamination of foods and feeds with Ochratoxin A (OTA) is a global problem, and its detoxification is challenging. In this study, Bacillus velezensis IS-6 culture isolate supernatant degraded 1.5 g/mL OTA by 89% after 24 h of incubation at 37 °C, whereas viable cells and intra-cell extracts were less effective. The OTA degradation by B. velezensis IS-6 was an enzymatic process mediated by the culture supernatant. The degradation activity was optimal at 37 °C and pH 7.0, and Fe2+ and Cu2+ ions enhanced the OTA degradation. The LC-MS/MS analysis confirmed that structure of OTA was modified, resulting in the production of OTα that was less toxic than OTA. The transcriptomic analysis of B. velezensis IS-6 showed that 38 differentially expressed genes (DEGs) were significantly up-regulated, and 24 DEGs were down-regulated after treatment with OTA. A novel OTA degradation enzyme Nudix hydrolase Nh-9 was successfully cloned and characterized from the up-regulated genes. The recombinant Nh-9 enzyme was overexpressed in Escherichia coli BL21 and purified by affinity chromatography, exhibiting 68% degradation activity against 1.0 µg/mL OTA at 37 °C in 24 h. The degraded product by the Nh-9 enzyme was identified as the less toxic OTα by LC-MS/MS. According to the findings, it can be inferred that Nh-9 is the main OTA-degrading enzyme in B. velezensis IS-6. Furthermore, OTA may be co-degraded by Nh-9, carboxylesterase, signal peptidase, and other degrading agents that are yet to be discovered in this strain.

Metagenome Analysis Identifies Microbial Shifts upon Deoxynivalenol Exposure and Post-Exposure Recovery in the Mouse Gut.

Deoxynivalenol (DON) is one of the most prevalent food-associated mycotoxins, and is known to cause a variety of adverse health effects on human and animals. Upon oral exposure, the intestine is the main target organ of DON. The current study unraveled that DON exposure (2 mg/kg bw/day or 5 mg/kg bw/day) can significantly reshape the gut microbiota in a mouse model. The study characterized the specific gut microbial strains and genes changed after DON exposure and also investigated the recovery of the microbiota upon either 2 weeks daily prebiotic inulin administration or 2 weeks recovery without intervention after termination of DON exposure (spontaneous recovery). The results obtained reveal that DON exposure causes a shift in gut microorganisms, increasing the relative abundance of Akkermansia muciniphila, Bacteroides vulgatus, Hungatella hathewayi, and Lachnospiraceae bacterium 28-4, while the relative abundance of Mucispirillum schaedleri, Pseudoflavonifractor sp. An85, Faecalibacterium prausnitzii, Firmicutes bacterium ASF500, Flavonifractor plautii, Oscillibacter sp. 1-3, and uncultured Flavonifractor sp. decreased. Notably, DON exposure enhanced the prevalence of A. muciniphila, a species considered as a potential prebiotic in previous studies. Most of the gut microbiome altered by DON in the low- and high-dose exposure groups recovered after 2 weeks of spontaneous recovery. Inulin administration appeared to promote the recovery of the gut microbiome and functional genes after low-dose DON exposure, but not after high-dose exposure, at which changes were exacerbated by inulin-supplemented recovery. The results obtained help to better understand the effect of DON on the gut microbiome, and the gut microbiota's recovery upon termination of DON exposure.

Accurate and non-destructive monitoring of mold contamination in foodstuffs based on whole-cell biosensor array coupling with machine-learning prediction models.

Mold contamination in foodstuffs causes huge economic losses, quality deterioration and mycotoxin production. Thus, non-destructive and accurate monitoring of mold occurrence in foodstuffs is highly required. We proposed a novel whole-cell biosensor array to monitor pre-mold events in foodstuffs. Firstly, 3 volatile markers ethyl propionate, 1-methyl-1 H-pyrrole and 2,3-butanediol were identified from pre-mold peanuts using gas chromatography-mass spectrometry. Together with other 3 frequently-reported volatiles from Aspergillus flavus infection, the volatiles at subinhibitory concentrations induced significant but differential response patterns from 14 stress-responsive Escherichia coli promoters. Subsequently, a whole-cell biosensor array based on the 14 promoters was constructed after whole-cell immobilization in calcium alginate. To discriminate the response patterns of the whole-cell biosensor array to mold-contaminated foodstuffs, optimal classifiers were determined by comparing 6 machine-learning algorithms. 100 % accuracy was achieved to discriminate healthy from moldy peanuts and maize, and 95 % and 98 % accuracy in discriminating pre-mold stages for infected peanuts and maize, based on random forest classifiers. 83 % accuracy was obtained to separate moldy peanuts from moldy maize by sparse partial least square determination analysis. The results demonstrated high accuracy and practicality of our method based on a whole-cell biosensor array coupling with machine-learning classifiers for mold monitoring in foodstuffs.

Microbial Enzymes Involved in the Biotransformation of Major Mycotoxins.

Mycotoxins, the most researched biological toxins, can contaminate food and feed, resulting in severe health implications for humans and animals. Physical, chemical, and biological techniques are used to mitigate mycotoxin contamination. The biotransformation method using whole microbial cells or isolated enzymes is the best choice to mitigate mycotoxins. Using specific enzymes may avoid the disadvantages of utilizing a full microbe, such as accidental harm to the product's organoleptic characteristics and hazardous safety features. Moreover, the degradation rates of the isolated enzymes are higher than those of the whole-cell reactions, and they are substrate-specific. Their specificity is comprehensive and is shown at the positional and/or chiral center in many circumstances. Currently, only a few enzymes of microbial origin are commercially available. Therefore, there is a need to identify more novel enzymes of microbial origin that can mitigate mycotoxins. In this review, we conducted an in-depth summary of the microbial enzymes involved in the biotransformation of mycotoxins.

Impact of environmental factors on ochratoxin A: From natural occurrence to control strategy.

Ochratoxin A (OTA) contamination and the associated issues of food security, food safety and economic loss are widespread throughout the world. The occurrence of OTA depends on ochratoxigenic fungi, foodstuffs and their environment. In this review, natural occurrence and control strategy of OTA, with a focus on the impact of environmental factors, are summarized. First, this manuscript introduces potentially contaminated foodstuffs, including the emerging ones which are not regulated in international legislation. Secondly, it gives an update of native producers based on foodstuffs and OTA biosynthesis. Thirdly, complicated environmental regulation is disassembled into individual factors in order to clarify their regulatory effect and mechanism. Finally, to emphasize control OTA at all stages of foodstuffs from farm to table, strategies used at crop planting, harvest, storage and processing stages are discussed.

Genome-Wide Identification and Expression Analysis of the Basic Leucine Zipper (bZIP) Transcription Factor Gene Family in Fusarium graminearum.

The basic leucine zipper (bZIP) is a widely found transcription factor family that plays regulatory roles in a variety of cellular processes including cell growth and development and various stress responses. However, the bZIP gene family has not been well studied at a genome-wide scale in Fusarium graminearum (Fg), a potent pathogen of cereal grains. In the present study, we conducted a genome-wide identification, characterization, and expression profiling of 22 F. graminearum bZIP (FgbZIP) genes at different developmental stages and under various abiotic stresses. All identified FgbZIPs were categorized into nine groups based on their sequence similarity and phylogenetic tree analysis. Furthermore, the gene structure analysis, conserved motif analysis, chromosomal localization, protein network studies, and synteny analysis were performed. The symmetry of the exon and intron varied with the phylogenetic groups. The post-translational modifications (PTMs) analysis also predicted several phosphorylation sites in FgbZIPs, indicating their functional diversity in cellular processes. The evolutionary study identified many orthogroups among eight species and also predicted several gene duplication events in F. graminearum. The protein modeling indicated the presence of a higher number of α-helices and random coils in their structures. The expression patterns of FgbZIP genes showed that 5 FgbZIP genes, including FgbZIP_1.1, FgbZIP_1.3, FgbZIP_2.6 FgbZIP_3.1 and FgbZIP_4.3, had high expression at different growth and conidiogenesis stages. Similarly, eight genes including FgbZIP_1.1, FgbZIP_1.6, FgbZIP_2.3, FgbZIP_2.4, FgbZIP_4.1, FgbZIP_4.2, FgbZIP_4.3 and FgbZIP_4.6 demonstrated their putative role in response to various abiotic stresses. In summary, these results provided basic information regarding FgbZIPs which are helpful for further functional analysis.