laeA Gene Research Articles

The isolation of sclerotinin A as an anti-malarial compound by utilization of a global secondary metabolism regulator, laeA gene

Previously, we successfully introduced laeA gene into a fungal strain in order to significantly increase the production of a bioactive compound, allowing use to discover novel biological activity. To demonstrate the universal applicability of the laeA gene introduction strategy for taping the potential of fungal secondary metabolism, in this present study, we created a library of microorganisms which we had the laeA gene inserted, and from that library we aimed to isolate compounds which are produced at significantly greater quantities compared to the respective wild type strains. From this investigation, we were able to isolate sclerotinin A (1) from Pochonia sp. KTF-0504 strain. We revealed that 1 showed anti-malarial activity against Plasmodium falciparum parasite strains. On the other hands, 1 showed no anti-fungal activity against multidrug-sensitive budding yeast. Our study implies that the utilization of the laeA gene in fungi is a versatile method for the discovery of drug candidates.

Histone H2B lysine 122 and lysine 130, as the putative targets of Penicillium oxalicum LaeA, play important roles in asexual development, expression of secondary metabolite gene clusters, and extracellular glycoside hydrolase synthesis.

Core histones in the nucleosome are subject to a wide variety of posttranslational modifications (PTMs), such as methylation, phosphorylation, ubiquitylation, and acetylation, all of which are crucial in shaping the structure of the chromatin and the expression of the target genes. A putative histone methyltransferase LaeA/Lae1, which is conserved in numerous filamentous fungi, functions as a global regulator of fungal growth, virulence, secondary metabolite formation, and the production of extracellular glycoside hydrolases (GHs). LaeA's direct histone targets, however, were not yet recognized. Previous research has shown that LaeA interacts with core histone H2B. Using S-adenosyl-L-methionine (SAM) as a methyl group donor and recombinant human histone H2B as the substrate, it was found that Penicillium oxalicum LaeA can transfer the methyl groups to the C-terminal lysine (K) 108 and K116 residues in vitro. The H2BK108 and H2BK116 sites on recombinant histone correspond to P. oxalicum H2BK122 and H2BK130, respectively. H2BK122A and H2BK130A, two mutants with histone H2B K122 or K130 mutation to alanine (A), were constructed in P. oxalicum. The mutants H2BK122A and H2BK130A demonstrated altered asexual development and decreased extracellular GH production, consistent with the findings of the laeA gene deletion strain (ΔlaeA). The transcriptome data showed that when compared to wild-type (WT) of P. oxalicum, 38 of the 47 differentially expressed (fold change ≥ 2, FDR ≤ 0.05) genes that encode extracellular GHs showed the same expression pattern in the three mutants ΔlaeA, H2BK122A, and H2BK130A. The four secondary metabolic gene clusters that considerably decreased expression in ΔlaeAalso significantly decreased in H2BK122A or H2BK130A. The chromatin of promotor regions of the key cellulolytic genes cel7A/cbh1 and cel7B/eg1 compacted in the ΔlaeA, H2BK122A, and H2BK130A mutants, according to the results of chromatin accessibility real-time PCR (CHART-PCR). The chromatin accessibility index dropped. The histone binding pocket of the LaeA-methyltransf_23 domain is compatible with particular histone H2B peptides, providing appropriate electrostatic and steric compatibility to stabilize these peptides, according to molecular docking. The findings of the study demonstrate that H2BK122 and H2BK130, which are histone targets of P. oxalicum LaeA in vitro, are crucial for fungal conidiation, the expression of gene clusters encoding secondary metabolites, and the production of extracellular GHs.

Re-discovery of MS-347a as a fungicide candidate through a new drug discovery platform with a multidrug-sensitive Saccharomyces cerevisiae screening system and the introduction of a global secondary metabolism regulator, laeA gene.

We found that the culture broth of fungi showed anti-fungal activity against multidrug-sensitive budding yeast. However, we could not identify the anti-fungal compound due to the small quantity. Therefore, we attempted to increase the productivity of the target compound by the introduction of a global secondary metabolism regulator, laeA to the strain, which led to the successful isolation of 10-folds greater amount of MS-347a (1) than Aspergillus sp. FKI-5362. Compound 1 was not effective against Candida albicans and the detailed anti-fungal activity of 1 remains unverified. After our anti-fungal activity screening, 1 was found to inhibit the growth of broad plant pathogenic fungal species belonging to the Ascomycota. It is noteworthy that 1 showed little insecticidal activity against silkworms, suggesting its selective biological activity against plant pathogenic fungi. Our study implies that the combination strategy of multidrug-sensitive yeast and the introduction of laeA is useful for new anti-fungal drug discovery.

PrlaeA Affects the Production of Roquefortine C, Mycophenolic Acid, and Andrastin A in Penicillium roqueforti, but It Has Little Impact on Asexual Development.

The regulation of fungal specialized metabolism is a complex process involving various regulators. Among these regulators, LaeA, a methyltransferase protein originally discovered in Aspergillus spp., plays a crucial role. Although the role of LaeA in specialized metabolism has been studied in different fungi, its function in Penicillium roqueforti remains unknown. In this study, we employed CRISPR-Cas9 technology to disrupt the laeA gene in P. roqueforti (PrlaeA) aiming to investigate its impact on the production of the specialized metabolites roquefortine C, mycophenolic acid, and andrastin A, as well as on asexual development, because they are processes that occur in the same temporal stages within the physiology of the fungus. Our results demonstrate a substantial reduction in the production of the three metabolites upon disruption of PrlaeA, suggesting a positive regulatory role of LaeA in their biosynthesis. These findings were further supported by qRT-PCR analysis, which revealed significant downregulation in the expression of genes associated with the biosynthetic gene clusters (BGCs) responsible for producing roquefortine C, mycophenolic acid, and andrastin A in the ΔPrlaeA strains compared with the wild-type P. roqueforti. Regarding asexual development, the disruption of PrlaeA led to a slight decrease in colony growth rate, while conidiation and conidial germination remained unaffected. Taken together, our results suggest that LaeA positively regulates the expression of the analyzed BGCs and the production of their corresponding metabolites in P. roqueforti, but it has little impact on asexual development.

Application of 6-Pentyl-α-Pyrone in the Nutrient Solution Used in Tomato Soilless Cultivation to Inhibit Fusarium oxysporum HF-26 Growth and Development

Soilless cultivation has increased in China. Like vegetables or ornamental plants, tomatoes can be cultivated in soilless culture systems. Fusarium wilt (FW) is an economic tomato disease. The nature and amount of volatile organic compounds in Trichoderma asperellum PT-15 were investigated, and 6-pentyl-α-pyrone (6-PP) was detected. Furthermore, the effect of 6-PP on Fusarium oxysporum HF-26 was evaluated. Results revealed that 25 mg/L 6-PP was the optimal concentration inhibiting F. oxysporum HF-26 and that the content of fusaric acid decreased considerably compared to that of the control. FUB1, FUB4, and FUB10 toxin synthesis and transport genes were downregulated. Additionally, VelA, velB, and LaeA genes were downregulated, reducing F. oxysporum mycelial growth and hyphae formation. 6-PP was added to the soilless culture solution in a greenhouse experiment. The results showed that an antifungal–nutrient solution containing 25 mg/L 6-PP significantly suppressed FW with 70.71% efficacy and a 27.23% disease index (DI), which were higher efficacy and lower DI than that of the control. Furthermore, treatment with an antifungal–nutrient solution containing 6-PP increased the levels of defence enzymes 24 h post-inoculation (hpi) compared with those at other time periods. The relative expression levels of the PR1, NPR1, PR2, and PR5 genes were considerably upregulated at 24 hpi.

A methyltransferase LaeA regulates ganoderic acid biosynthesis in Ganoderma lingzhi.

The methyltransferase LaeA is a global regulator involved in the biosynthesis of secondary metabolites by ascomycete fungi. However, little is known of its regulatory role in basidiomycete fungi. In this study, the laeA gene was identified in the basidiomycete Ganoderma lingzhi and its function in regulating the biosynthesis of anti-tumor ganoderic acids was evaluated. A laeA deletion (ΔlaeA) Ganoderma strain exhibited significantly reduced concentration of ganoderic acids. qRT-PCR analysis further revealed that the transcription levels of genes involved in the biosynthesis of ganoderic acids were drastically lower in the ΔlaeA strain. Moreover, deletion of laeA resulted in decreased accumulation of intermediates and abundances of asexual spores in liquid static culture of G. lingzhi. In contrast, constitutive overexpression of laeA resulted in increased concentration of ganoderic acids. These results demonstrate an essential role of LaeA in the regulation of ganoderic acid biosynthesis in Ganoderma.

Disruption of the Chitin Biosynthetic Pathway Results in Significant Changes in the Cell Growth Phenotypes and Biosynthesis of Secondary Metabolites of Monascus purpureus.

In this study, the gene monascus-5162 from Monascus purpureus LQ-6, identified as chitin synthase gene VI (chs6), was knocked out to disrupt the chitin biosynthetic pathway and regulate the biosynthesis of Monascus pigments (MPs) and citrinin. The results showed that the aerial hyphae on a solid medium were short and sparse after the deletion of chs6 in M. purpureus LQ-6, significantly reducing the germination percentage of active spores to approximately 22%, but the colony diameter was almost unaffected. Additionally, the deletion of chs6 changed the mycelial morphology of M. purpureus LQ-6 during submerged fermentation and increased its sensitivity to environmental factors. MP and citrinin biosynthesis was dramatically inhibited in the recombinant strain. Furthermore, comparative transcriptome analysis revealed that the pathways related to spore development and growth, including the MAPK signaling pathway, chitin biosynthetic pathway, and regulatory factors LaeA and WetA genes, were significantly downregulated in the early phase of fermentation. The mRNA expression levels of genes in the cluster of secondary metabolites were significantly downregulated, especially those related to citrinin biosynthesis. This is the first detailed study to reveal that chs6 plays a vital role in regulating the cell growth and secondary metabolism of the Monascus genus.

Assessing the efficacy of chitosan nanomatrix incorporated with Cymbopogon citratus (DC.) Stapf essential oil against the food-borne molds and aflatoxin B1 production in food system

The chitosan nanomatrix incorporated with Cymbopogon citratus essential oil (Ne-CcEO) possess enhanced efficacy against the food-borne molds and aflatoxin B1 production compared to free essential oil. The CcEO was encapsulated inside the chitosan nanomatrix with an average size 147.41 ± 16.18 nm and characterized by Scanning electron microscopy, Fourier transforms infrared spectroscopy, and X-ray diffraction assay. The encapsulation efficiency and loading capacity were ranged between (41.68–76.78%) and (5.3–8.80%). The biochemical and in-silico analysis results revealed the interference in functioning of membrane integrity, mitochondrial membrane potential, antioxidant defense, carbon source metabolism, methylglyoxal, and laeA gene in response to treatment of Ne-CcEO (0.5 μl/ml). In addition, Ne-CcEO significantly protects the deterioration of Pennisetum glaucum (L.) R. Br. seed samples by A. flavus, aflatoxin B1 contamination, and lipid peroxidation. The Ne-CcEO could be considered as promising antifungal additives for the control of food-borne molds and aflatoxin B1 contamination in the food system.

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

Overexpression of global regulator LaeA increases secondary metabolite production in Monascus purpureus.

Monascus is a filamentous fungus that produces several secondary metabolites. Here, we investigated the effects of the global regulator LaeA on the synthesis of pigments and monacolin K in Monascus purpureus with spectrophotometer and HPLC methods. The LaeA gene was isolated from M. purpureus M1 to create an overexpression construct. An LaeA-overexpressing strain L3 was with 48.6% higher monacolin K production than the M1 strain. The L3 strain also produced higher Monascus pigments than the M1 strain. SEM showed that LaeA overexpression resulted in altered mycelial morphology. Compared with the M1 strain, the L3 strain expressed higher levels of monacolin K synthesis-related genes mokA, mokB, mokE, and mokH. Overall, these results suggest that LaeA plays a role in regulating the production of secondary metabolites and mycelial growth in Monascus. This study provides important insights into the mechanisms underlying the effects of the LaeA gene on the secondary metabolites of M. purpureus.

Essential oils trigger an antifungal and anti-aflatoxigenic effect on Aspergillus flavus via the induction of apoptosis-like cell death and gene regulation

In the present study, we offer detailed insights into the mechanisms of action of clove and rosemary essential oils (EOs) on Aspergillus flavus and provide evidence that the induction of apoptosis-like cell death and the downregulation of metabolic genes trigger their antifungal and anti-aflatoxigenic effects. The mode of action of both EOs at the cellular level was investigated through the detection of early (nuclear condensation) and late apoptosis (damage of plasma membrane) in hyphae exposed to EOs and a growth rate assay. Furthermore, we investigated changes at the molecular level by relative gene expression analysis and determined the expression of genes related to secondary metabolism (laeA) and mechanisms of virulence (lipA and metP) in A. flavus. Additionally, the inhibition of aflatoxin B1 production by both EOs was detected by high-performance liquid chromatography. The results indicated that the exposure of A. flavus to clove and rosemary EOs induced significant apoptosis-like cell death and the downregulation of the laeA, lipA and metP genes, thereby providing novel insights into the cellular and molecular antifungal mechanisms of EOs against mycotoxigenic species.

The Role of LaeA and LovE Regulators in Lovastatin Biosynthesis with Exogenous Polyamines in Aspergillus terreus

The filamentous fungus Aspergillus terreus is the main industrial producer of a cholesterol-lowering drug, lovastatin, and simvastatin obtained on its basis. The biosynthesis of lovastatin in A. terreus is under the control of two major positive regulators, the LovE pathway-specific regulator and LaeA global regulator of the secondary metabolism of fungi. It is shown that laeA expression can be negatively regulated by LovE, the Zn2Cys6 transcription factor of lovastatin biosynthesis. The overexpression of lovE under the control of the gpdA constitutive promoter from Aspergillus nidulans, which results in a 10- to 30-fold increase in lovastatin production is accompanied by a decrease in laeA expression in the fermentation process. The observed negative interrelationship between the LovE and LaeA regulators consists of the downregulation of lov resistance and transport genes in A. terreus with an additional copy of lovE and phenotypically manifests itself as polyamine toxicity for lovastatin production. Polyamine addition into the medium in the course of fermentation of A. terreus OE::lovE leads to a decrease in the lovastatin production by 30–40% and inhibited the expression of the lov and laeA genes.

1,3-Diaminopropane and Spermidine Upregulate Lovastatin Production and Expression of Lovastatin Biosynthetic Genes in Aspergillus terreus via LaeA Regulation

The filamentous fungus Aspergillus terreus is the main industrial producer of lovastatin, a cholesterol-lowering drug that is also used as a semiproduct for simvastatin production. The exogenous addition of such polyamines as 1,3-diaminopropane or spermidine during the fermentation of wild-type A. terreus ATCC 20542 and overproducing A. terreus 43-16 strains results in a 20−45% increase in lovastatin production. In the case of strain 43-16, the maximum production level (10 g/L) was observed three days earlier than in the control variant. During this fermentation period, the expression level of genes belonging to the lovastatin biosynthetic cluster and the laeA gene (a global regulator of a fungal secondary metabolism) are increased, but the expression patterns for genes of the polyamine metabolic pathway were similar for both strains.

Apple Intrinsic Factors Modulating the Global Regulator, LaeA, the Patulin Gene Cluster and Patulin Accumulation During Fruit Colonization by Penicillium expansum.

The mycotoxin patulin is produced in colonized tissue by Penicillium expansum during storage of apples and is significantly affected by environmental factors that contribute to its accumulation. Few reports have, however, examined the effect of natural intrinsic factors associated with the fruit on the production of patulin. Here, we find that with advancing maturity, Golden Delicious apples show increased concentrations of total soluble solids (TSS) from 14 to 17% associated with the increased expression of the global transcription factor involved in regulation of secondary metabolite biosynthesis in filamentous fungi, laeA expression and patulin accumulation. However, the apple cultivar Granny Smith, with similar TSS values but differing in pH levels and malic acid concentrations, showed reduced expression levels of laeA and the patulin biosynthesis gene cluster (pat genes) and patulin accumulation, suggesting a complexity of host factors contribution to patulin accumulation during P. expansum colonization. To start elucidating these apple intrinsic factors, we examined their in vitro impact on laeA and pat gene expression concomitant with patulin synthesis. Increasing sucrose concentrations from 15 to 175 mM repressed laeA and pat gene expression and patulin production. However, this affect was modified and often reversed and sometimes accentuated by changes in pH, or the addition of malic acid or the major apple phenolic compounds, chlorogenic acid and epicatechin. While the increase in malic acid from 0 to 1% increased laeA and pat gene expression, the decrease in pH from 3.5 to 2.5 reduced their expression. Also the increased laeA and pat genes expressions at increasing epicatechin concentrations from 0 to 1 mM, was reversed by increasing sucrose concentrations, all together suggesting the complexity of the interactions in vivo.

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion.

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

Identification and characterization of genes involved in kojic acid biosynthesis in Aspergillus flavus

Although Aspergillus flavus is a strong producer strain for kojic acid (KA), up till now there is no report on genes encoding KA. Conversely, KA genes from A. oryzae have been reported, with kojA, kojR and kojT genes, an enzyme gene, a transcription factor and a transporter, respectively, having been cloned and characterized. Thus, in the present study, we aimed to identify and characterize KA genes from A. flavus. To achieve this goal, we first produced by gamma irradiation A. flavus and A. oryzae mutants with improved levels of KA with respect to wild type (WT) strains. Then, we cloned orthologs of A. oryzae kojA, kojR and kojT from A. flavus. Finally, we analyzed the expression levels of KA genes in mutant (MT) and WT strains of both species. The mutants A. flavus HAk1-M2 and A. oryzae HAk2-M26 were found to produce KA at levels 2.5- and 3.02-fold higher than their parent strains after 7–10 days under shaking conditions. The cloned A. flavus genes AFLA_096040, AFLA_096050 and AFLA_096060, exhibited 99–100% similarity to kojA, kojR and kojT genes from A. oryzae, respectively. Gene expression analyses revealed that the expression levels of KA genes nicely overlap with KA levels both in A. flavus and A. oryzae, MT and WT strains. In addition, we cloned the laeA gene, which controls the biosynthesis of secondary metabolites from A. flavus. The results showed that, unlike A. oryzae, the expression of laeA in A. flavus does not correlate with KA biosynthesis. Our findings are of importance and reveal that the gene cluster comprising AFLA_096040, AFLA_096050 and AFLA_096060 is potentially involved in KA biosynthesis.

Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in Penicillium chrysogenum: cross-talk regulation of secondary metabolite pathways

Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.

LaeA and VeA are involved in growth morphology, asexual development, and mycotoxin production in Alternaria alternata

Alternaria alternata is a common filamentous fungus that contaminates various fruits, grains and vegetables causing important economic losses to farmers and the food industry. A. alternata is a mycotoxigenic mould, which may jeopardize human and animal health. Two of the most common A. alternata mycotoxins found in food and feed are alternariol and alternariol monomethyl ether. In this study we examined the role of LaeA and VeA, two regulatory proteins belonging to the velvet family, which have been described to be involved in several functions in many fungi including secondary metabolism. We found that deletion of laeA and veA genes, respectively, greatly reduced sporulation and strongly compromised mycotoxin production, both in vitro or during pathogenesis of tomato fruits. We have also studied how the loss of laeA and veA may affect expression of genes related to alternariol and alternariol monomethyl ether biosynthesis (pksJ and altR), and to melanin biosynthesis (cmrA, pksA).

Expression of Genes by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus Isolated from Brazil Nuts.

The aims of the present study were to monitor the production of aflatoxin B1 (AFB1) and mycelial growth, and to evaluate the expression of genes directly and indirectly involved in the biosynthesis of aflatoxins by Aspergillus flavus isolated from Brazil nuts. Six previously identified A. flavus strains were grown on coconut agar at 25°C for up to 10 days. Mycotoxins were separated by high-performance liquid chromatography and fungal growth was measured daily using the diametric mycelial growth rate. Transcriptional analysis was performed by real-time polymerase chain reaction (PCR) after 2 and 7 d of incubation using specific primers (aflR, aflD, aflP, lipase, metalloprotease, and LaeA). Three (50%) of the six A. flavus isolates produced AFB1 (ICB-1, ICB-12, and ICB-54) and three (50%) were not aflatoxigenic (ICB-141, ICB-161, and ICB-198). Aflatoxin production was observed from d 2 of incubation (1.5 ng/g for ICB-54) and increased gradually with time of incubation until d 10 (15,803.6 ng/g for ICB-54). Almost all A. flavus isolates exhibited a similar gene expression pattern after 2 d of incubation (p > 0.10). After 7 d of incubation, the LaeA (p < 0.05) and metalloprotease (p < 0.05) genes were the most expressed by nonaflatoxigenic strains, whereas aflatoxigenic isolates exhibited higher expression of the aflR (p < 0.05) and aflD genes (p < 0.05). Our results suggest that the expression of aflR and aflD is correlated with aflatoxin production in A. flavus and that overexpression of aflR could affect the transcriptional and aflatoxigenic pattern (ICB-54). Elucidation of the molecular mechanisms that regulate the secondary metabolism of toxigenic fungi may permit the rational silencing of the genes involved and consequently the programmed inhibition of aflatoxin production. Knowledge of the conditions, under which aflatoxin genes are expressed, should contribute to the development of innovative and more cost-effective strategies to reduce and prevent aflatoxin contamination in Brazil nuts.

Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites.

The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations.