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.     

Nitrogen availability and production of bikaverin and gibberellins in Gibberella fujikuroi

Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the nitrogen source, but not after depletion of phosphate. Despite this similarity, the regulation of both pathways by nitrogen involved two different mechanisms. This conclusion was supported by the fact that the production of bikaverin, in contrast to the gibberellins, was not inhibited by nitrate in a mutant that could not utilize it. The different regulation of both pathways was clearly demonstrated by a mutant that overproduced bikaverin but lacked gibberellins. An optimal bikaverin production required a low pH, with a sharp drop at about pH 5. The syntheses of fungal secondary metabolites, such as bikaverin and gibberellins, are not subject to common regulation, but respond to various combinations of signals, such as nitrogen availability, nitrate and the pH of the medium.     

Two novel classes of enzymes are required for the biosynthesis of aurofusarin in Fusarium graminearum

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.     

Biosynthesis of naphthoquinone pigments by fungi of the genus Fusarium

We studied biosynthesis of colored naphthoquinone metabolites by Fusarium decemcellulare, F. graminearum, and F. bulbigenum fungi. F. bulbigenum and F. graminearum synthesized bikaverin and aurofusarin, respectively, which depended on the conditions of cultivation. F. decemcellulare synthesized soluble extracellular naphthoquinones of the naphthazarin structure (javanicin, anhydrojavanicin, fusarubin, anhydrofusarubin, bostricoidin, and novarubin) or extracellular dimeric naphthoquinone aurofusarin. Biosynthesis of naphthoquinone pigments was shown to be the main fungal response to stress exposure. It occurs under conditions of growth inhibition or arrest.     

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 GACI 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 wildtype 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.     

Stimulation of bikaverin production by sucrose and by salt starvation in Fusarium fujikuroi

The fungus Fusarium fujikuroi (Gibberella fujikuroi mating group C) exhibits a rich secondary metabolism that includes the synthesis of compounds of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. The effect of the carbon source on their production was checked using a two-phase incubation protocol, in which nine different sugars were added upon transfer of the fungus from repressed to appropriate inducing conditions, i.e., nitrogen starvation for gibberellins and bikaverin and illumination for carotenoids production. Most of the carbon sources allowed the synthesis of these metabolites in significant amounts. However, bikaverin production was strongly increased by the presence of sucrose in comparison to other carbon sources, an effect not exhibited for the production of gibberellins and carotenoids. The bikaverin inducing effect was enhanced in the absence of phosphate and/or sulfate. Similar results were also observed in carotenoid-overproducing strains known to be altered in bikaverin production. The induction by salt starvation, but not by sucrose, correlated with an increase in messenger RNA levels of gene bik1, encoding a polyketide synthase of the bikaverin pathway.     

Specific regulatory interconnection between the leucine and histidine pathways of Neurospora crassa.

Leucine auxotrophs of Neurospora fall into two discrete categories with respect to sensitivity to the herbicide, 3-amino-1,2,4-triazole. The pattern of resistance corresponds exactly to the ability to produce the leucine pathway control elements, alpha-isopropylmalate and the leu-3 product. An analysis of the regulatory response of the production of enzymes of histidine biosynthesis to alpha-isopropylmalate implicates the control elements of the leucine pathway as important components of the mechanism governing the production of the target enzyme of aminotriazole inhibition, imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19). The evidence suggests that the regulatory interconnection between the two pathways is direct and is independent of other general integrating regulatory mechanisms which appear to be operative in both pathways. A general method for isolating leu-1 and leu-2, as well as other regulatory mutants, is described, which takes advantage of the specificity of the resistance to the inhibitor. Use of analogous systems is prescribed for the analysis of other regulatory interconnections which, like this one, might not be anticipated directly from structural or biosynthetic considerations.     

Impact of ammonium permeases mepA, mepB, and mepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi

In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of ΔmepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In ΔmepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as ΔmepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the ΔmepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi.     

Bikaverin production and applications

Bikaverin is a reddish pigment produced by different fungal species, most of them from the genus Fusarium, with antibiotic properties against certain protozoa and fungi. Chemically, bikaverin is a polyketide with a tetracyclic benzoxanthone structure, resulting from the activity of a specific class I multifunctional polyketide synthase and subsequent group modifications introduced by a monooxygenase and an O-methyltransferase. In some fungi, bikaverin is found with smaller amounts of a precursor molecule, called norbikaverin. Production of these metabolites by different fungal species depends on culture conditions, but it is mainly affected by nitrogen availability and pH. Regulation of the pathway has been investigated in special detail in the gibberellin-producing fungus Fusarium fujikuroi, whose genes and enzymes responsible for bikaverin production have been recently characterized. In this fungus, the synthesis is induced by nitrogen starvation and acidic pH, and it is favored by other factors, such as aeration, sulfate and phosphate starvation, or sucrose availability. Some of these inducing agents increase mRNA levels of the enzymatic genes, organized in a coregulated cluster. The biological properties of bikaverin include antitumoral activity against different cancer cell lines. The diverse biological activities and the increasing information on the biochemical and genetic basis of its production make bikaverin a metabolite of increasing biotechnological interest.     

Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor

Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.     

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.     

Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi

The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H(2)O(2)), but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.     

Biochemical pathways and mechanisms nitrogen, amino acid, and carbon metabolism

For both nitrogen and carbon metabolism there exist specific regulatory mechanisms to enable cells to assimilate a wide variety of nitrogen and carbon sources. Superimposed are regulatory circuits, the so called nitrogen and carbon catabolite regulation, to allow for selective use of “rich” sources first and “poor” sources later. Evidence points to the importance of specific regulatory mechanisms for short term adaptations, while generalized control circuits are used for long term modulation of nitrogen and carbon metabolism. Similarly a variety of regulatory mechanisms operate in amino acid metabolism. Modulation of enzyme activity and modulation of enzyme levels are the outstanding regulatory mechanisms. In prokaryotes, attenuation and repressor/operator control are predominant, besides a so called “metabolic control” which integrates amino acid metabolism into the overall nutritional status of the cells. In eukaryotic cells compartmentation of amino acid metabolites as well as of part of the pathways becomes an additional regulatory factor; pathway specific controls seem to be rare, but a complex regulatory network, the “general control of amino acid biosynthesis”, coordinates the synthesis of enzymes of a number of amino acid biosynthetic pathways.