Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

Three-Motif Molecular Junction Type Covalent Organic Frameworks for Efficient Photocatalytic Aerobic Oxidation.

Covalent organic frameworks (COFs), with the features of flexible structure regulation and easy introduction of functional groups, have aroused broad interest in the field of photocatalysis. However, due to the low light absorption intensity, low photoelectron conversion efficiency, and lack of suitable active sites, it remains a great challenge to achieve efficient photocatalytic aerobic oxidation reactions. Herein, based on reticular chemistry, we rationally designed a series of three-motif molecular junction type COFs, which formed dual photosensitizer coupled redox molecular junctions containing multifunctional COF photocatalysts. Significantly, due to the strong light adsorption ability of dual photosensitizer units and integrated oxidation and reduction features, the PY-BT COF exhibited the highest activity for photocatalytic aerobic oxidation. Especially, it achieved a photocatalytic benzylamine conversion efficiency of 99.9% in 2.5 h, which is much higher than that of the two-motif molecular junctions with only one photosensitizer or redox unit lacking COFs. The mechanism of selective aerobic oxidation was studied through comprehensive experiments and density functional theory calculations. The results showed that the photoinduced electron transfer occurred from PY and then through triphenylamine to BT. Furthermore, the thermodynamics energy for benzylamine oxidation on PY-BT COF was much lower than that for others, which confirmed the synergistic effect of dual photosensitizer coupled redox molecular junction COFs. This work provided a new strategy for the design of functional COFs with three-motif molecular junctions and also represented a new insight into the multifunctional COFs for organic catalytic reactions.

Structural and functional characterization of a new thermophilic-like OYE from Aspergillus flavus.

Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (AfOYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T1/2 of AfOYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, AfOYE1 exhibited high reduction activity in a wide pH range (pH 5.5-8.0). AfOYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, ß-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (S)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of AfOYE1 revealed that the "cap" region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal "finger" structure endow the catalytic cavity of AfOYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of AfOYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. KEY POINTS: A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T1/2 of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv.

Lipopeptide-mediated bacterial interaction enables cooperative predator defense.

Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a strong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.

AnAzf1 acts as a positive regulator of ochratoxin A biosynthesis in Aspergillus niger.

Aspergillus niger produces genotoxic and carcinogenic ochratoxin A (OTA) that severely threatens human and animal health. Transcription factor Azf1 is essential in regulating fungal cell development and primary metabolism. However, its effect and mechanism on secondary metabolism are unclear. Here, we characterized and deleted a Azf1 homolog gene, An15g00120 (AnAzf1), in A. niger, which completely blocked OTA production, and repressed the OTA cluster genes, p450, nrps, hal, and bzip at the transcriptional level. The results indicated that AnAzf1 was a positive regulator of OTA biosynthesis. Transcriptome sequencing results showed that the AnAzf1 deletion significantly upregulated antioxidant genes and downregulated oxidative phosphorylation genes. Enzymes involved in reactive oxygen species (ROS) scavenging, including catalase (CAT) and peroxidase (POD) were increased, and the corresponding ROS levels were decreased. Upregulation of genes (cat, catA, hog1, and gfd) in the MAPK pathway and downregulation of genes in iron homeostasis were associated with decreased ROS levels, linking the altered MAPK pathway and iron homeostasis to lower ROS levels caused by AnAzf1 deletion. Additionally, enzymes including complex I (NADH-ubiquinone oxidoreductase), and complex V (ATP synthase), as well as ATP levels, were significantly decreased, indicating impaired oxidative phosphorylation caused by the AnAzf1-deletion. During lower ROS levels and impaired oxidative phosphorylation, OTA was not produced in ∆AnAzf1. Together, these results strongly suggested that AnAzf1 deletion blocked OTA production in A. niger by a synergistic interference of ROS accumulation and oxidative phosphorylation. KEY POINTS: • AnAzf1 positively regulated OTA biosynthesis in A. niger. • Deletion of AnAzf1 decreased ROS levels and impaired oxidative phosphorylation. • An altered MAPK pathway and iron homeostasis were associated with lower ROS levels.

Inhibitory effect of (E)-2-heptenal on Aspergillus flavus growth revealed by metabolomics and biochemical analyses.

The prevention of fungal proliferation in postharvest grains is critical for maintaining grain quality and reducing mycotoxin contamination. Fumigation with natural gaseous fungicides is a promising and sustainable approach to protect grains from fungal spoilage. In this study, the antifungal activities of (E)-2-alkenals (C5-C10) on Aspergillus flavus were tested in the vapor phase, and (E)-2-heptenal showed the highest antifungal activity against A. flavus. (E)-2-Heptenal completely inhibited A. flavus growth at 0.0125 µL/mL and 0.2 µL/mL in the vapor phase and liquid contact, respectively. (E)-2-Heptenal can disrupt the plasma membrane integrity of A. flavus via leakage of intracellular electrolytes. Scanning electron microscopy indicated that the mycelial morphology of A. flavus was remarkably affected by (E)-2-heptenal. Metabolomic analyses indicated that 49 metabolites were significantly differentially expressed in A. flavus mycelia exposed to 0.2 µL/mL (E)-2-heptenal; these metabolites were mainly involved in galactose metabolism, starch and sucrose metabolism, the phosphotransferase system, and ATP-binding cassette transporters. ATP production was reduced in (E)-2-heptenal-treated A. flavus, and Janus Green B staining showed reduced cytochrome c oxidase activity. (E)-2-Heptenal treatment induced oxidative stress in A. flavus mycelia with an accumulation of superoxide anions and hydrogen peroxide and increased activities of superoxide dismutase and catalase. Simulated storage experiments showed that fumigation with 400 µL/L of (E)-2-heptenal vapor could completely inhibit A. flavus growth in wheat grains with 20% moisture; this demonstrates its potential use in preventing grain spoilage. This study provides valuable insights into understanding the antifungal effects of (E)-2-heptenal on A. flavus. KEY POINTS : • (E)-2-Heptenal vapor showed the highest antifungal activity against A. flavus among (C5-C10) (E)-2-alkenals. • The antifungal effects of (E)-2-heptenal against A. flavus were determined. • The antifungal actions of (E)-2-heptenal on A. flavus were revealed by metabolomics and biochemical analyses.

Transcriptomic and biochemical analyses revealed antifungal mechanism of trans-anethole on Aspergillus flavus growth.

Plant volatile compounds have great potential for preventing and controlling fungal spoilage in post-harvest grains. Recently, we have reported the antifungal effects of trans-anethole, the main volatile constituent of the Illicium verum fruit, on Aspergillus flavus. In this study, the inhibitory mechanisms of trans-anethole against the growth of A. flavus mycelia were investigated using transcriptomic and biochemical analyses. Biochemical and transcriptomic changes in A. flavus mycelia were evaluated after exposure to 0.2 µL/mL trans-anethole. Scanning electron microscopy showed that trans-anethole treatment resulted in the surface wrinkling of A. flavus mycelia, and calcofluor white staining confirmed that trans-anethole treatment disrupted the mycelial cell wall structure. Annexin V-fluorescein isothiocyanate/propidium iodide double staining suggested that trans-anethole induced apoptosis in A. flavus mycelia. Reduced mitochondrial membrane potential and DNA damage were observed in trans-anethole-treated A. flavus mycelia using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine and 4',6-diamidino-2-phenylindole staining, respectively. 2',7'- Dichloro-dihydro-fluorescein diacetate staining and biochemical assays demonstrated that trans-anethole treatment cause the accumulation of reactive oxygen species in the A. flavus mycelia. Transcriptome results showed that 1673 genes were differentially expressed in A. flavus mycelia exposed to trans-anethole, which were mainly associated with multidrug transport, oxidative phosphorylation, citric acid cycle, ribosomes, and cyclic adenosine monophosphate signaling. We propose that trans-anethole can inhibit the growth of A. flavus mycelia by disrupting the cell wall structure, blocking the multidrug transport process, disturbing the citric acid cycle, and inducing apoptosis. This study provides new insights into the inhibitory mechanism of trans-anethole on A. flavus mycelia and will be helpful for the development of natural fungicides. KEY POINTS: • Biochemical analyses of A. flavus mycelia exposed to trans-anethole were performed • Transcriptomic changes in trans-anethole-treated A. flavus mycelia were analyzed • An inhibitory mechanism of trans-anethole on the growth of A. flavus mycelia was proposed.

Protection of postharvest grains from fungal spoilage by biogenic volatiles.

Fungal spoilage of postharvest grains poses serious problems with respect to food safety, human health, and the economic value of grains. The protection of cereal grains from deleterious fungi is a critical aim in postharvest grain management. Considering the bulk volume of grain piles in warehouses or bins and food safety, fumigation with natural gaseous fungicides is a promising strategy to control fungal contamination on postharvest grains. Increasing research has focused on the antifungal properties of biogenic volatiles. This review summarizes the literature related to the effects of biogenic volatiles from microbes and plants on spoilage fungi on postharvest grains and highlights the underlying antifungal mechanisms. Key areas for additional research on fumigation with biogenic volatiles in postharvest grains are noted. The research described in this review supports the protective effects of biogenic volatiles against grain spoilage by fungi, providing a basis for their expanded application in the management of postharvest grains.

Dual Photosensitizer Coupled Three-Dimensional Metal-Covalent Organic Frameworks for Efficient Photocatalytic Reactions.

In this work, we innovatively assembled two types of traditional photosensitizers, that is pyridine ruthenium/ferrum (Ru(bpy)3 2+ /Fe(bpy)3 2+ ) and porphyrin/metalloporphyrin complex (2HPor/ZnPor) by covalent linkage to get a series of dual photosensitizer-based three-dimensional metal-covalent organic frameworks (3D MCOFs), which behaved strong visible light-absorbing ability, efficient electron transfer and suitable band gap for highly efficient photocatalytic hydrogen (H2 ) evolution. Rubpy-ZnPor COF achieved the highest H2 yield (30 338 µmol g-1 h-1 ) with apparent quantum efficiency (AQE) of 9.68 %@420 nm, which showed one of the best performances among all reported COF based photocatalysts. Furthermore, the in situ produced H2 was successfully tandem used in the alkyne hydrogenation with ≈99.9 % conversion efficiency. Theoretical calculations reveal that both the two photosensitizer units in MCOFs can be photoexcited and thus contribute optimal photocatalytic activity. This work develops a general strategy and shows the great potential of using multiple photosensitive materials in the field of photocatalysis.

Relative Local Electron Density Tuning in Metal-Covalent Organic Frameworks for Boosting CO2 Photoreduction.

The high local electron density and efficient charge carrier separation are two important factors to affect photocatalytic activity, especially for the CO2 photoreduction reaction. However, the systematic studies on the structure-functional relationship regarding the above two factors based on precisely structure model are rarely reported. Herein, as a proof-of-concept, we developed a new strategy on the evaluation of local electron density by controlling the relative electron-deficient (ED) and electron-rich (ER) intensity of monomer at a molecular level based on three rational-designed vinylene-linked sp2 carbon-covalent organic frameworks (COFs). As expected, the as-prepared vinylene-linked sp2 carbon-conjugated metal-covalent organic framework (MCOFs) (VL-MCOF-1) with molecular junction exhibited excellent activities for CO2 -to-HCOOH conversion (283.41 µmol g-1 h-1 ) and high selectivity of 97.1 %, much higher than the VL-MCOF-2 and g-C34 N6 -COF, which is due to the synergistic effect of the multi-electronic metal clusters (Cu3 (PyCA)3 ) (PyCA=pyrazolate-4-carboxaldehyde) as strong ER roles and cyanopyridine units as ED roles and active sites, as well as the boosted photo-induced charge separation efficiency of vinyl connection and increased light utilization ability. These results not only provide a strategy for regulating the electron-density distribution of photocatalysts at the molecular level but also offers profound insights for metal clusters-based COFs to effective CO2 conversion.

Lysine 2-hydroxyisobutyrylation orchestrates cell development and aflatoxin biosynthesis in Aspergillus flavus.

Lysine 2-hydroxyisobutyrylation (Khib ) is a recently identified post-translational modification (PTM) that regulates numerous cellular metabolic processes. In pathogenic microorganism, although glycolysis and fungal virulence are regulated by Khib , its potential roles in fungi remain to be elusive. Our preliminary results showed that levels of Khib fluctuate over time in Aspergillus flavus, which frequently contaminates crops and produces carcinogenic aflatoxins. However, the perception of Khib function in A. flavus is limited, especially in mycotoxin-producing strains. Here, we performed a comprehensive analysis of Khib in A. flavus, and 7156 Khib sites were identified in 1473 proteins. Notably, we demonstrated that Khib of AflM, a key enzyme in aflatoxin biosynthesis, affected conidia production and sclerotia formation. Furthermore, aflM deletion impaired aflatoxin biosynthesis, and more importantly, strains in which Khib was mimicked by K to T mutation at K49, K179 and K180 sites showed reduced aflatoxin production compared with wild type and ΔaflM complementation strains. These results indicate that Khib at these sites of AflM negatively regulates aflatoxin biosynthesis in A. flavus. In summary, our study revealed the potential roles of Khib in A. flavus, and particularly shed light on a new way to regulate aflatoxin production via Khib .

The antifungal mechanisms of plant volatile compound 1-octanol against Aspergillus flavus growth.

The exploitation of active ingredients from plant volatile organic compounds as natural gaseous fungicides shows remarkable potential for controlling fungal decay in postharvest agroproducts. Although 1-octanol is a common component of cereal volatiles, its antifungal potency against spoilage fungi in postharvest grains remains unclear. In this study, we studied the effectiveness of 1-octanol against Aspergillus flavus growth in postharvest grains and its mechanisms of action. 1-Octanol vapor and liquid contact dose-dependently inhibited A. flavus spore germination and mycelial growth at a low concentration. The simulated storage experiment demonstrated that 300 µL/L of 1-octanol vapor completely controlled A. flavus growth in wheat, corn, and paddy grains with 20% moisture content. 1-Octanol treatment irreversibly damaged the conidial and mycelial morphology of A. flavus and caused electrolyte leakage due to reduced plasma membrane integrity. It induced apoptosis along with morphological abnormalities, phosphatidylserine externalization, mitochondrial membrane potential depolarization, intracellular reactive oxygen species accumulation, and DNA fragmentation in A. flavus cells. Metabolomic analysis revealed that 1-octanol treatment disrupted the biosynthesis of unsaturated fatty acids, ATP-binding cassette transporters, amino acid metabolism, and glycerophospholipid metabolism. This study demonstrated the promising application potential of 1-octanol as a biofumigant for preventing fungal spoilage of postharvest cereal grains. KEY POINTS: • (1) 1-Octanol inhibits Aspergillus flavus growth in the vapor phase and liquid contact; • (2) 1-Octanol damages membrane integrity and induces apoptosis of A. flavus; • (3) Metabolomic changes in A. flavus mycelia were analyzed after 1-octanol treatment.

Transcriptomics analyses and biochemical characterization of Aspergillus flavus spores exposed to 1-nonanol.

The exploitation of plant volatile organic compounds as biofumigants to control postharvest decaying of agro-products has received considerable research attention. Our previous study reported that 1-nonanol, the main constituent of cereal volatiles, can inhibit Aspergillus flavus growth and has the potential as a biofumigant to control the fungal spoilage of cereal grains. However, the antifungal mechanism of 1-nonanol against A. flavus is still unclear at the molecular level. In this study, the minimum inhibitory concentration and minimum fungicidal concentration of 1-nonanol against A. flavus spores were 2 and 4 µL/mL, respectively. Scanning electron microscopy revealed that the 1-nonanol can distort the morphology of A. flavus spore. Annexin V-FITC/PI double staining showed that 1-nonanol induced phosphatidylserine eversion and increased membrane permeability of A. flavus spores. Transcriptional profile analysis showed that 1-nonanol treatment mainly affected the expression of genes related to membrane damage, oxidative phosphorylation, blockage of DNA replication, and autophagy in A. flavus spores. Flow cytometry analysis showed that 1-nonanol treatment caused hyperpolarization of mitochondrial membrane potential and accumulation of reactive oxygen species in A. flavus spores. 4',6-diamidino-2-phenylindole staining showed that treatment with 1-nonanol destroyed the DNA. Biochemical analysis results confirmed that 1-nonanol exerted destructive effects on A. flavus spores by decreasing intracellular adenosine triphosphate content, reducing mitochondrial ATPase activity, accumulating hydrogen peroxide and superoxide anions, and increasing catalase and superoxide dismutase enzyme activities. This study provides new insights into the antifungal mechanisms of 1-nonanol against A. flavus. KEY POINTS: • 1-Nonanol treatment resulted in abnormal morphology of A. flavus spores. • 1-Nonanol affects the expression of key growth-related genes of A. flavus. • The apoptosis of A. favus spores were induced after exposed to 1-nonanol.

Mechanisms underlying the inhibitory effects of linalool on Aspergillus flavus spore germination.

Biogenic volatile organic compounds hold remarkable potential for controlling fungal decay in agro- and food products. Recently, we reported that linalool, the major volatile component of the Zanthoxylum schinifolium pericarp, showed great potential as a biofumigant to control Aspergillus flavus growth in postharvest grains. In this study, the inhibitory effects of linalool on A. flavus growth in stored grains and its underlying mechanism were investigated through transcriptomic and biochemical analyses. Linalool vapor at 800 µL/L can effectively prevent A. flavus growth in 22% moisture wheat grains. Linalool at 2 µL/mL completely inhibited the germination of A. flavus spores, and 10 µL/mL caused spore death. Scanning electron microscopy revealed that linalool treatment caused wrinkling and spore breakage. Transcriptomics showed that 3806 genes were significantly differentially expressed in A. flavus spores exposed to 2 µL/mL linalool, predominantly showing enrichment regarding the ribosome, DNA replication, glutathione metabolism, peroxisome, and MAPK signaling pathways. Flow cytometry showed that linalool treatment caused hyperpolarization of mitochondrial membrane potential. 4,6-Diamidino-2-phenylindole staining indicated that linalool caused DNA fragmentation in A. flavus spores, and monodansylcadaverine staining confirmed that linalool induced autophagy in A. flavus spores. We thus propose that linalool can damage the plasma membrane, cause mitochondrial dysfunction and DNA damage, and induce autophagy in A. flavus spores. These findings considerably improve our understanding of the mechanisms underlying the inhibitory effects of linalool on A. flavus, which is crucial regarding the development of applications to prevent postharvest grain spoilage due to A. flavus infestations. KEY POINTS: • The inhibitory potency of linalool on A. flavus spore germination was determined. • Transcriptomic analyses were performed to identify differentially expressed genes of A. flavus exposed to linalool. • A functional mechanism underlying the inhibitory effects of linalool on A. flavus spore germination is proposed.

Transcriptome analysis reveals the underlying mechanism of heptanal against Aspergillus flavus spore germination.

Methods of controlling Aspergillus flavus contamination in agro-products have attracted attention because of its impact on global food security. We previously reported that the natural cereal volatile heptanal could effectively inhibit A. flavus growth and showed great potential as a bio-preservative agent. In this study, the minimum inhibitory concentration and minimum fungicide concentration of heptanal could change the surface morphology of A. flavus spores, causing them to wrinkle and collapse. Transcriptomic analysis showed that heptanal treatment significantly changed the expression of several genes involved in cell wall and plasma damage, reactive oxygen species (ROS) accumulation, energy metabolism, AMPK-activated protein kinase, biosynthesis of unsaturated fatty acids, RNA degradation, and DNA replication. Heptanal-induced early apoptosis of A. flavus spores was characterized by decreased mitochondrial membrane potential, increased intracellular ROS production, and DNA fragmentation. This study provides new insight into the inhibitory mechanism of heptanal against A. flavus and points to its potential application as a bio-preservative. KEY POINTS: • Heptanal can effectively inhibit A. flavus growth in cereal grains. • The transcriptional changes in A. flavus spores exposed to heptanal were analyzed. • The antifungal mechanism of heptanal against A. flavus was elucidated.

Antifungal mechanism of 1-nonanol against Aspergillus flavus growth revealed by metabolomic analyses.

Chemical control of fungal spoilage of postharvest cereal grains is an important strategy for the management of grain storage. Here, the potential antifungal activity of 1-nonanol, a main component of cereal volatiles, against Aspergillus flavus was studied. The growth of A. flavus was completely inhibited by 0.11 and 0.20 µL/mL 1-nonanol at vapor and liquid contact phases, respectively. Metabolomic analysis identified 135 metabolites whose expression was significantly different between 1-nonanol-treated and untreated A. flavus. These metabolites were involved in the tricarboxylic acid cycle, amino acid biosynthesis, protein degradation and absorption, aminoacyl-tRNA biosynthesis, mineral absorption, and in interactions with ABC transporters. Biochemical validation confirmed the disruptive effect of 1-nonanol on A. flavus growth, as indicated by the leakage of intracellular electrolytes, decreased succinate dehydrogenase, mitochondrial dehydrogenase, and ATPase activity, and the accumulation of reactive oxygen species. We speculated that 1-nonanol could disrupt cell membrane integrity and mitochondrial function and might induce apoptosis of A. flavus mycelia. Simulated grain storage experiments showed that 1-nonanol vapor, at a concentration of 264 µL/L, completely inhibited A. flavus growth in wheat, corn, and paddy grain with an 18% moisture content. This study provides new insights into the antifungal mechanism of 1-nonanol against A. flavus, indicating that it has a promising potential as a bio-preservative to prevent fungal spoilage of postharvest grains. KEY POINTS: • 1-Nonanol showed higher antifungal activity against A. flavus. • The antifungal mechanisms of 1-nonanol against A. flavus were revealed. • 1-Nonanol could damage cell membrane integrity and mitochondrial function.

Hexanal induces early apoptosis of Aspergillus flavus conidia by disrupting mitochondrial function and expression of key genes.

Aspergillus flavus is a notorious saprophytic fungus that compromises the quantity and quality of postharvest grains and produces carcinogenic aflatoxins. The natural compound hexanal disrupts cell membrane synthesis and mitochondrial function and induces apoptosis in A. flavus; here, we investigated the molecular mechanisms underlying these effects. The minimum inhibition and fungicidal concentration (MIC and MFC) of hexanal against A. flavus spores were 3.2 and 9.6 µL/mL, respectively. Hexanal exposure resulted in abnormal spore morphology and early spore apoptosis. These changes were accompanied by increased reactive oxygen species production, reduced mitochondrial membrane potential, and DNA fragmentation. Transcriptomic analysis revealed that hexanal treatment greatly altered the metabolism of A. flavus spores, including membrane permeability, mitochondrial function, energy metabolism, DNA replication, oxidative stress, and autophagy. This study provides novel insights into the mechanism underlying the antifungal activity of hexanal, suggesting that hexanal can be used an anti-A. flavus agent for agricultural applications. KEY POINTS: • Hexanal exposure resulted in abnormal spore morphology. • The apoptotic characteristics of A. flavus were induced after hexanal treatment. • Hexanal could change the expression of key A. flavus growth-related genes.

Metabolomic analyses revealed multifaceted effects of hexanal on Aspergillus flavus growth.

Hexanal, a natural volatile organic compound, exerts antifungal activity against Aspergillus flavus; however, the mechanisms underlying these effects are unclear. In this study, we found that the growth of A. flavus mycelium was completely inhibited following exposure to 0.4 µL/mL hexanal (minimal inhibitory concentration). A detailed metabolomics survey was performed to identify changes in metabolite production by A. flavus cells after exposure to 1/2 the minimal inhibitory concentration of hexanal for 6 h, which revealed significant differences in 70 metabolites, including 20 upregulated and 50 downregulated metabolites. Among them, levels of L-malic acid, α-linolenic acid, phosphatidylcholine, D-ribose, riboflavin, D-mannitol, D-sorbitol, and deoxyinosine were significantly reduced. The metabolomics results suggest that the metabolites are mainly involved in the tricarboxylic acid cycle (TCA), ABC transport system, and membrane synthesis in A. flavus cells. Hexanal treatment reduced succinate dehydrogenase and mitochondrial dehydrogenase activity and stimulated superoxide anion and hydrogen peroxide accumulation in A. flavus mycelia. Increases in the electric conductivity and A260nm of the culture supernatant indicated cell membrane leakage. Therefore, hexanal appears to disrupt cell membrane synthesis, induce mitochondrial dysfunction, and increase oxidative stress in A. flavus mycelia. KEY POINTS: • Metabolite changes of A. flavus mycelia were identified after hexanal treatment. • Most differential metabolites were downregulated in hexanal-treated A. flavus. • An antifungal model of hexanal against A. flavus was proposed.

Sub3 inhibits Aspergillus flavus growth by disrupting mitochondrial energy metabolism, and has potential biocontrol during peanut storage.

BACKGROUND: Aspergillus flavus, a saprophytic fungus, is regularly detected in oil-enriched seeds. During colonization, this organism releases aflatoxins that pose a serious risk to food safety and human health. Therefore, an eco-friendly biological approach to inhibit the pathogen is desirable. RESULTS: Experimental results indicated that A. flavus spores could not germinate in potato dextrose broth culture medium, when the concentration of Sub3 exceeded 0.15 g L-1 . Morphological evaluation performed by flow cytometry and scanning electron microscopy indicated that spores were shrunken and pitted following Sub3 exposure. Physiological assessment using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide, 2,7-dichlorodihydrofluorescein diacetate and 4',6-diamidino-2-phenylindole staining revealed damaged cell membranes, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species levels, and elevated large nuclear condensation and DNA fragmentation. Moreover, mitochondrial dehydrogenase activity was reduced by 29.42% and 45.48% after treatment with 0.1 and 0.15 g L-1 Sub3, respectively. Additionally, colonization capacity in peanut was significantly decreased, and the number of spores on seeds treated with Sub3 was decreased by 26.86% (0.1 g L-1 ) and 77.74% (0.15 g L-1 ) compared with the control group. CONCLUSION: Sub3 likely inhibits A. flavus by crossing the cell wall and targeting the cell membrane, disrupting mitochondrial energy metabolism, and inducing DNA damage, leading to spore death. Thus, Sub3 may provide a useful biocontrol strategy to control A. flavus growth in peanuts. © 2020 Society of Chemical Industry.

Pleiotropic regulation of daptomycin synthesis by DptR1, a LuxR family transcriptional regulator.

Daptomycin, produced by Streptomyces roseosporus is a novel cyclic lipopeptide antibiotic for treatment of Gram-positive bacteria caused infections. While, the regulatory mechanism of daptomycin synthesis has not been fully understood. Here we reported that DptR1, a LuxR family transcriptional regulator, played a pleiotropic regulatory role on daptomycin synthesis, for the first time. Deletion or over-expressing of dptR1 decreases the daptomycin's production, increases the transcriptional levels of the core dpt genes of day 3 and decreases the transcriptional levels of the core dpt genes of day 4, sharply, which indicates the transcriptional regulation of DptR1 on daptomycin synthesis is complex and time-ordered. The transcriptional levels of dptR2 increase in dptR1 deletion mutant (DR1), but decrease in dptR1 over-expression mutant (OR1), dramatically, compared to the starting strain of Streptomyces roseosporus N3 (WT), on the 3rd day, which indicates that DptR1 represses the transcription of dptR2. While, the transcriptional levels of dptR3 both in DR1 and OR1 decrease obviously, compared to WT, on the 3rd and 4th day. Comparative analysis of promoters' activities, using xylE gene as the reporter, showed that DptR1 activated the transcription of its own gene of dptR1 and represses the transcription of the dptR3 by affecting the promoter activities. While DptR1 may affect the expression of dptR2 indirectly, not by affecting the promoter activity of dptR2. DptR1, a LuxR family transcriptional regulator, played a pleiotropic regulation role on daptomycin synthesis.