Bikaverin Biosynthesis Research Articles

FvatfA regulates growth, stress tolerance as well as mycotoxin and pigment productions in Fusarium verticillioides

FvatfA from the maize pathogen Fusarium verticillioides putatively encodes the Aspergillus nidulans AtfA and Schizasaccharomyces pombe Atf1 orthologous bZIP-type transcription factor, FvAtfA. In this study, a ΔFvatfA deletion mutant was constructed and then genetically complemented with the fully functional FvatfA gene. Comparing phenotypic features of the wild-type parental, the deletion mutant and the restored strains shed light on the versatile regulatory functions played by FvAtfA in (i) the maintenance of vegetative growth on Czapek-Dox and Potato Dextrose agars and invasive growth on unwounded tomato fruits, (ii) the preservation of conidiospore yield and size, (iii) the orchestration of oxidative (H2O2, menadione sodium bisulphite) and cell wall integrity (Congo Red) stress defences and (iv) the regulation of mycotoxin (fumonisins) and pigment (bikaverin, carotenoid) productions. Expression of selected biosynthetic genes both in the fumonisin (fum1, fum8) and the carotenoid (carRA, carB) pathways were down-regulated in the ΔFvatfA strain resulting in defected fumonisin production and considerably decreased carotenoid yields. The expression of bik1, encoding the polyketide synthase needed in bikaverin biosynthesis, was not up-regulated by the deletion of FvatfA meanwhile the ΔFvatfA strain produced approximately ten times more bikaverin than the wild-type or the genetically complemented strains. The abolishment of fumonisin production of the ΔFvatfA strain may lead to the development of new-type, biology-based mycotoxin control strategies. The novel information gained on the regulation of pigment production by this fungus can be interesting for experts working on new, Fusarium-based biomass and pigment production technologies.Key points• FvatfA regulates vegetative and invasive growths of F. verticillioides.• FvatfA also orchestrates oxidative and cell wall integrity stress defenses.• The ΔFvatfA mutant was deficient in fumonisin production.• FvatfA deletion resulted in decreased carotenoid and increased bikaverin yields.

Putative metabolic pathway for the bioproduction of bikaverin and intermediates thereof in the wild Fusarium oxysporum LCP531 strain

Fungal naphthoquinones, like red bikaverin, are of interest due to their growing applications in designing pharmaceutical products. Though considerable work has been done on the elucidation of bikaverin biosynthesis pathway in Fusarium fujikuroi, very few reports are available regarding its bioproduction in F. oxysporum. We are hereby proposing a putative metabolic pathway for bikaverin bioproduction in a wild F. oxysporum strain by cross-linking the pigment profiles we obtained under two different fermentation conditions with literature. Naphthoquinone pigments were extracted with a pressurized liquid extraction method, and characterized by HPLC–DAD and UHPLC-HRMS. The results led to the conclusions that the F. oxysporum LCP531 strain was able to produce bikaverin and its various intermediates, e.g., pre-bikaverin, oxo-pre-bikaverin, dinor-bikaverin, me-oxo-pre-bikaverin, and nor-bikaverin, in submerged cultures in various proportions. To our knowledge, this is the first report of the isolation of these five bikaverin intermediates from F. oxysporum cultures, providing us with steady clues for confirming a bikaverin metabolic pathway as well as some of its regulatory patterns in the F. oxysporum LCP531 strain, based on the previously reported model in F. fujikuroi. Interestingly, norbikaverin accumulated along with bikaverin in mycelial cells when the strain grew on simple carbon and nitrogen sources and additional cofactors. Along bikaverin production, we were able to describe the excretion of the toxin beauvericin as main extrolite exclusively in liquid medium containing complex nitrogen and carbon sources, as well as the isolation of ergosterol derivate in mycelial extracts, which have potential for pharmaceutical uses. Therefore, culture conditions were also concluded to trigger some specific biosynthetic route favoring various metabolites of interest. Such observation is of great significance for selective production of pigments and/or prevention of occurrence of others (aka mycotoxins).

Conserved Responses in a War of Small Molecules between a Plant-Pathogenic Bacterium and Fungi.

ABSTRACTSmall-molecule signaling is one major mode of communication within the polymicrobial consortium of soil and rhizosphere. While microbial secondary metabolite (SM) production and responses of individual species have been studied extensively, little is known about potentially conserved roles of SM signals in multilayered symbiotic or antagonistic relationships. Here, we characterize the SM-mediated interaction between the plant-pathogenic bacterium Ralstonia solanacearum and the two plant-pathogenic fungi Fusarium fujikuroi and Botrytis cinerea. We show that cellular differentiation and SM biosynthesis in F. fujikuroi are induced by the bacterially produced lipopeptide ralsolamycin (synonym ralstonin A). In particular, fungal bikaverin production is induced and preferentially accumulates in fungal survival spores (chlamydospores) only when exposed to supernatants of ralsolamycin-producing strains of R. solanacearum. Although inactivation of bikaverin biosynthesis moderately increases chlamydospore invasion by R. solanacearum, we show that other metabolites such as beauvericin are also induced by ralsolamycin and contribute to suppression of R. solanacearum growth in vitro. Based on our findings that bikaverin antagonizes R. solanacearum and that ralsolamycin induces bikaverin biosynthesis in F. fujikuroi, we asked whether other bikaverin-producing fungi show similar responses to ralsolamycin. Examining a strain of B. cinerea that horizontally acquired the bikaverin gene cluster from Fusarium, we found that ralsolamycin induced bikaverin biosynthesis in this fungus. Our results suggest that conservation of microbial SM responses across distantly related fungi may arise from horizontal transfer of protective gene clusters that are activated by conserved regulatory cues, e.g., a bacterial lipopeptide, providing consistent fitness advantages in dynamic polymicrobial networks.

Signaling Governed by G Proteins and cAMP Is Crucial for Growth, Secondary Metabolism and Sexual Development in Fusarium fujikuroi

The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs). In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s) exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus.

The cAMP Signaling Pathway in Fusarium verticillioides Is Important for Conidiation, Plant Infection, and Stress Responses but Not Fumonisin Production

Fusarium verticillioides is one of the most important fungal pathogens of maize. Mycotoxin, fumonisins produced by this pathogen pose a threat to human and animal health. Because cAMP signaling has been implicated in regulating diverse developmental and infection processes in fungal pathogens, in this study, we aimed to elucidate the function of the cAMP-protein kinase A (PKA) pathway in toxin production and plant infection in F. verticillioides. Targeted deletion mutants were generated for the CPK1 and FAC1 genes that encode a catalytic subunit of PKA and the adenylate cyclase, respectively. Defects in radial growth and macroconidiation were observed in both the cpk1 and fac1 deletion mutants. The fac1 mutant also was significantly reduced in virulence and microconidiation but increased in tolerance to heat and oxidative stresses. These phenotypes were not observed in the cpk1 mutant, indicating that additional catalytic subunit of PKA must exist and function downstream from FAC1. The fac1 mutant formed microconidia mainly in false heads. The expression levels of the hydrophobin genes HYD1 and HYD2, which are known to be associated with change in formation of microconidia, were significantly reduced in the fac1 mutant. Expression of F. verticillioides GSY2 and HSP26 genes, two other putative downstream targets of FAC1, was increased in the fac1 mutant and may be associated with its enhanced stress tolerance. Although fumonisin production was normal, biosynthesis of bikaverin was increased in the fac1 mutant, suggesting that FAC1 and cAMP signaling may have pathway-or metabolite-specific regulatory roles in secondary metabolism. Overall, the pleiotropic defects of the fac1 deletion mutant indicate that the cAMP-PKA pathway is involved in growth, conidiation, bikaverin production, and plant infection in F. verticillioides.

Regulation of Carotenogenesis and Secondary Metabolism by Nitrogen in Wild-Type Fusarium fujikuroi and Carotenoid-Overproducing Mutants

The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.

GAC1, a gene encoding a putative GTPase-activating protein, regulates bikaverin biosynthesis in Fusarium verticillioides

Fusarium verticillioides (teleomorph Gibberella moniliformis) is an ascomycete known to produce a variety of secondary metabolites, including fumonisins, fusaric acid and bikaverin. These metabolites are synthesized when the fungus is under stress, notably nutrient limitations. To date we have limited understanding of the complex regulatory process associated with fungal secondary metabolism. In this study we generated a collection of F. verticillioides mutants by using REMI (restriction enzyme mediated integration) mutagenesis and in the process identified a strain, R647, that carries a mutation in a gene designated GAC1. Mutation in the GAC1 locus, which encodes a putative GTPase activating protein, resulted in the increased production of bikaverin, suggesting that GAC1 is negatively associated with bikaverin biosynthesis. Complementation of R647 with the wild-type GAC1 gene restored the bikaverin production level to that of the wild-type progenitor, demonstrating that gac1 mutation was directly responsible for the overproduction of bikaverin. We also demonstrated that AREA, encoding global nitrogen regulator, and PKS4, encoding polyketide synthase, are downstream genes that respectively are regulated positively and negatively by GAC1. Our results suggest that GAC1 plays an important role in signal transduction regulating bikaverin production in F. verticillioides.

Role of the Fusarium fujikuroi TOR Kinase in Nitrogen Regulation and Secondary Metabolism

In Fusarium fujikuroi, the biosynthesis of gibberellins (GAs) and bikaverin is under control of AreA-mediated nitrogen metabolite repression. Thus far, the signaling components acting upstream of AreA and regulating its nuclear translocation are unknown. In Saccharomyces cerevisiae, the target of rapamycin (TOR) proteins, Tor1p and Tor2p, are key players of nutrient-mediated signal transduction to control cell growth. In filamentous fungi, probably only one TOR kinase-encoding gene exists. However, nothing is known about its function. Therefore, we investigated the role of TOR in the GA-producing fungus F. fujikuroi in order to determine whether TOR plays a role in nitrogen regulation, especially in the regulation of GA and bikaverin biosynthesis. We cloned and characterized the F. fujikuroi tor gene. However, we were not able to create knockout mutants, suggesting that TOR is essential for viability. Inhibition of TOR by rapamycin affected the expression of AreA-controlled secondary metabolite genes for GA and bikaverin biosynthesis, as well as genes involved in transcriptional and translational regulation, ribosome biogenesis, and autophagy. Deletion of fpr1 encoding the FKBP12-homologue confirmed that the effects of rapamycin are due to the specific inhibition of TOR. Interestingly, the expression of most of the TOR target genes has been previously shown to be also affected in the glutamine synthetase mutant, although in the opposite way. We demonstrate here for the first time in a filamentous fungus that the TOR kinase is involved in nitrogen regulation of secondary metabolism and that rapamycin affects also the expression of genes involved in translation control, ribosome biogenesis, carbon metabolism, and autophagy.

Deletion of the Gibberella fujikuroi glutamine synthetase gene has significant impact on transcriptional control of primary and secondary metabolism.

In Gibberella fujikuroi, the gibberellin (GA) and bikaverin biosynthesis are under control of nitrogen metabolite repression. However, the signalling components acting upstream of AREA are still unknown. We investigated the role of glutamine synthetase (GS) both as an enzyme and as a possible regulator in the nitrogen regulation system. We cloned and replaced the GS-encoding gene, glnA-Gf. The mutants grow with a phenotype different from the wild type in the presence of glutamine. They were unable to express nitrogen-repressed GA and bikaverin biosynthetic genes even under nitrogen starvation conditions. Complementation with the glnA-Gf wild-type copy fully restored GS activity, the expression of secondary metabolism genes, and the production of GAs and the red pigment, bikaverin. In order to find more target genes of GS, differential cDNA-screening and differential hybridization of macroarrays were performed using cDNA from the wild type and DeltaglnA mutant as probes. Several genes were dramatically up- or downregulated in the mutant. Among them are genes involved in N- and C-catabolism, and in transcriptional and translation control. Some of these genes are also under AREA control. Treatment with the GS inhibitor l-methionine sulphoximine resulted in similar expression patterns as in the glnA mutant with ammonium as nitrogen source, whereas glutamine can overcome the up- or downregulation of most but not all of the target genes. These findings suggest that not only glutamine, but also GS itself might play an important role in nitrogen metabolite repression.

The polyketide synthase gene pks4 from Gibberella fujikuroi encodes a key enzyme in the biosynthesis of the red pigment bikaverin

The ascomycete Gibberella fujikuroi mating population C (MP-C) is well known for the production of gibberellins, but also produces many other secondary metabolites, including the red polyketide pigment bikaverin. Here, we used a differential display method to clone a polyketide synthase gene pks4 responsible for the first step of bikaverin biosynthesis. Sequence analysis indicated that pks4 encoded a 2009-amino acid polypeptide consisting of four functional domains: β-ketoacyl synthase (KS), acyltransferase (AT), acyl carrier (ACP), and thioesterase (TE). Disruption of pks4 resulted in the loss of both pks4 transcripts and bikaverin biosynthesis in G. fujikuroi cultures. Expression of pks4 is strongly repressed by high amounts of ammonium and basic pH. Unexpectedly, pks4 was overexpressed in mutants of the regulatory gene, areA, which is responsible for the activation of nitrogen assimilation genes. Three additional polyketide synthase genes have been cloned from G. fujikuroi MP-C by heterologous hybridization. The presence of these four PKS genes demonstrates the diversity of polyketide biosynthetic pathways in this fungus.

Do the pigments differ chemotaxonomically fungi belonging to genusFusarium?

Biosynthesis of bikaverin - red pigment responsible for characteristic colour ofFusarium cultures and known as vacuolation and aging factor - by 60Fusarium isolates belonging to 9 species was examined. We observed that bikaverin is a common metabolite produced by species of Liseola, Mar tiella, Elegans and Dlaminiella sections.Twenty nine isolates of fourFusarium species (F. moniliforme, F. subglutinans, F. proliferatum andF. anthophilum), belonging to the following Liseola section were investigated on their ability to biosynthesis of secondary metabolites bikaverin, 8-methylfusarubin, 3,8-dimethylfusarubin, 8-methylbostrycoidin, nectriafurone and fusarin C. All examined isolates of section Liseola formed bikaverin and 8-methylfusarubin, while 21 isolates additionally produced 8-methylbostrycoidin. OnlyF. moniliforme isolates were able to form fusarin C. We did not observe chemotaxonomical differences betweenFusarium, species according their ability to biosynthesis of such secondary metabolites as bikaverin, 8-methylfusarubin and 8-methylbostrycoidin.

Biosynthesis of bikaverin in Fusarium oxysporum. Use of 13C nuclear magnetic resonance with homonuclear 13C decoupling to locate adjacent 13C labels.

Bikaverin obtained by supplementing cultures of Fusarium oxysporum with singly and doubly 13C labeled acetate was enriched by approximately 0.5 atom percent with the 13C isotope. At this low enrichment 13C NMR spectra of samples labeled from (1-13C)- and (2-13C) acetate did not show, unequivocally, the pattern of isotopic incorporation. Small sample size, poor solubility and difficulties in the assignment of resonances also restricted the amount of information thacetate. The difficulty was overcome by using 13C homonuclear single-frequency decoupling in conjunction with 1H heteronuclear decoupling to locate bonded 13C-13C pairs. The carbon skeleton of bikaverin was shown to be biosynthesized entirely by condensation of acetate units and the pattern of assembly was established.