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.     

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.     

Gibberellins and Carotenoids in the Wild Type and Mutants of Gibberella fujikuroi

A new screening procedure was used to isolate 14 gib mutants of Gibberella fujikuroi with modifications in the production of gibberellins. The production of carotenoids and gibberellins was investigated in the gib mutants and in representative car mutants with various modifications of carotenoid biosynthesis. The determinations of gibberellins were carried out with a simplified fluorescence method. One of the mutants lacked both gibberellins and carotenoids. In many mutants the two pathways compensated each other: an increase in the production of one group of compounds was accompanied by a decrease in the production of the other. Under certain conditions the compensation was quantitative when the output of the two pathways was measured in moles of the common precursor, geranylgeranyl pyrophosphate. α-Picoline, an inhibitor of lycopene cyclase in G. fujikuroi, inhibits gibberellin biosynthesis. Other agents that affect the accumulation of carotenoids have no noticeable effect on the accumulation of gibberellins; such is the case with diphenylamine and β-ionone, two inhibitors of phytoene dehydrogenation, and visible light, which stimulates carotenogenesis.     

Nutritional regulation of proteinase production in the fungus, Tritirachium album

The fungus, Tritirachium album, produces a number of proteinases under proper conditions. We have studied the nutritional regulation mechanisms for proteinase production in the mold, i.e. the effects of carbon and nitrogen sources, and the influence of starvation. Proteinase production was induced when the nitrogen source was an exogenous protein or peptide, such as peptones or yeast extract. The production rate was affected by the amount of available inducing substrate. Inorganic nitrogen compounds, i.e., ammonium or nitrate salts, had a repressing effect on the production. Production was not induced if a detectable concentration of glucose or sucrose was present in the medium. Starvation did not trigger proteinase production. Journal of Industrial Microbiology & Biotechnology (2000) 24, 369–373. Received 20 January 1999/ Accepted in revised form 06 March 2000     

Gibberellin Biosynthesis: Genetic Studies in Gibberella fujikuroi

Biochemical genetic studies on the production of gibberellins by the fungus Gibberella fujikuroi have identified two genes which control different steps in the biosynthetic pathway. One gene (g₁) controls the production of all of the gibberellins: the second gene (g₂) controls the production of GA₁ and GA₃ only. Ascospores are not ordered in the ascus of this fungus. This apparent spore slippage precludes mapping of these genes to their respective centromeres.     

Bikaverin,an antibiotic fromGibberella fujikuroi,effective againstLeishmania brasiliensis

The red quinone C₂₀H₁₄O₈ (mp 322–324), named bikaverin was isolated from mycelium ofGibberella fujikuroi. Bikaverin is specifically effective againstLeishmania brasiliensis while is not effective against other protozoa, bacteria and fungi. For production of bikaverin in a submerged culture the most convenient media are those with a natural source of nitrogen (corn-steep liquor, soyabean meal) in which the content of bikaverin ranged from 1.0 to 1.7% of dry weight of mycelium.     

Selective production of bikaverin in a fluidized bioreactor with immobilized Gibberella fujikuroi

The best culture medium composition for the production of bikaverin by Gibberella fujikuroi in shake-flasks, i.e. 100 g glucose l^–1; 1 g NH₄Cl l^–1; 2 g rice flour l^–1; 5 g KH₂PO₄ l^–1 and 2.5 g MgSO₄ l^–1, was obtained through a fractional factorial design and then scaled-up to a fluidized bioreactor. The effects of carbon and nitrogen concentrations, inoculum size, aeration, flow rate and bead sizes on batch bikaverin production using immobilized G. fujikuroi in a fluidized bioreactor were determined by an orthogonal experimental design. Concentrations of up to 6.83 g bikaverin l^–1 were obtained when the medium contained 100 g glucose l^–1 and 1 g NH₄Cl l^–1 with an inoculum ratio of 10% v/v, an aeration rate of 3 volumes of air per volume of medium min^–1, and a bead size of 3 mm. Based on dry weight, the bikaverin production was 30–100 times larger than found in submerged culture and approximately three times larger than reported for solid substrate fermentation.     

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.     

Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

     

Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal

The ability of the fungus Fusarium oxysporum to solubilize lignite was found to depend on the presence of a specific carbon source. When grown on glucose or another carbohydrate, the fungus is unable to solubilize coal but it produces the red dye bikaverin. In the coal-solubilizing state, which can be induced by cultivation in the presence of glutamate or gluconate, the fungus does not produce bikaverin. The presence or absence of the pigment can therefore be taken as an indicator of the ability of the fungus to solubilize coal. Addition of extracted and purified bikaverin to F. oxysporum growing on glutamate or gluconate inhibits coal solubilization. Hence, F. oxysporum offers a suitable system for investigating the mechanism of microbial coal degradation by comparing the two growth-substrate-controlled physiological states.     

Mathematical description of bikaverin production in a fluidized bed bioreactor

Summary  Growth of Gibberella fujikuroi in submerged cultures occurs as micelles or filamentous hyphae dispersed in fluid and pellets or stable, spherical agglomerations. Gibberella fujikuroi growth, substrate consumption and bikaverin production kinetics obtained from submerged batch fermentation were fitted to three different sigmoid models: two and three-parameter Gompertz models and one Logistic model. Growth fitting was used to compare between models and select the best one by means of an F test. The best model for describing growth was the two-parameter Gompertz model and was used for glucose consumption and bikaverin production fitting. Data from eight different schemes of fermentations were analysed and parameter estimation was carried out by means of minimization of residual sum of squares. Some characteristic values obtained with the two-parameter Gompertz model fit are: μ=0.028 h⁻¹, Y_x/s=0.1089 g substrate/g biomass, α =0.1384 g product/g biomass.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immobilized growing cells of Gibberella fujikuroi P-3 for production of gibberellic acid and pigment in batch and semi-continuous cultures

Summary The performance of immobilized growing cells of Gibberella fujikuroi P-3 was affected by the immobilization agent used, nature and age of cells, mycelial cell density, size of beads and inclusion of linseed oil. The beads, prepared by using standardized procedures, could be used for 8 cycles without affecting productivity in semicontinuous culture. The rate of production of gibberellic acid was 0.58–0.66 mg/l/h. An inverted conical fluidized bioreactor, based on the design employed in continuous plant cell culture, was adopted. This bioreactor offers many advantages. The pigment produced by the fungus is not similar to bikaverin, norbikaverin and O-dimethylanhydrofusarubin, the known polyketides.     

Pectin lyase production by Penicillium griseoroseum: Effect of tea extract, caffeine, yeast extract, and pectin

Summary Mycelial growth and production of extracellular pectin lyase by Penicillium griseoroseum at different concentrations of inducers were investigated. The fungus was cultured in mineral medium using sucrose as a carbon source and caffeine, yeast extract, tea extract or pectin as inducers. Caffeine, yeast extract and tea extract in the presence of sucrose, and tea extract alone were capable of inducing pectin lyase in P. griseoroseum, even at low concentrations.     

Biosynthesis and beneficial effects of microbial gibberellins on crops for sustainable agriculture

Soil microbes promote plant growth through several mechanisms such as secretion of chemical compounds including plant growth hormones. Among the phytohormones, auxins, ethylene, cytokinins, abscisic acid and gibberellins are the best understood compounds. Gibberellins were first isolated in 1935 from the fungus Gibberella fujikuroi and are synthesized by several soil microbes. The effect of gibberellins on plant growth and development has been studied, as has the biosynthesis pathways, enzymes, genes and their regulation. This review revisits the history of gibberellin research highlighting microbial gibberellins and their effects on plant health with an emphasis on the early discoveries and current advances that can find vital applications in agricultural practices.     

Inductive effect produced by a mixture of carbon source in the production of gibberellic acid by Gibberella fujikuroi

Gibberellic acid has been known since 1954 but its effect on rice still remains very important in the agricultural world. Gibberellic acid (GA₃) is the main secondary metabolite produced by the Gibberella fujikuroi fungus. This hormone is of great importance in agriculture and the brewing industry, due to its fast and strong effects at low concentrations (μg) on the processes of growth stimulation, flowering, stem elongation, and germination of seeds, among others. Plant promoters of growth production such as the gibberellins, especially the GA₃ are a priority in obtaining better harvests in the agricultural area and by extension, improving the food industry. Three routes to obtaining GA₃ have been reported: extraction from plants, chemical synthesis and microbial fermentation. The latter being the most common method used to produce GA₃. In this investigation, glucose-corn oil mixture was used as a carbon source on the basis of 40 g of carbon in a 7 L stirred tank bioreactor. A pH of 3.5, 29°C, 600 min⁻¹ agitation and 1 vvm aeration were maintained and controlled with a biocontroller connected to the bioreactor, throughout the entire culture time. The carbon source mixture affected the fermentation time as well as the production of the GAs. The production of 380 mg GA₃L⁻¹ after 288 h of fermentation was obtained when the glucose-corn oil mixture was employed contrasting the 136 mg GA₃L⁻¹ at 264 h of culture when only glucose was used.     

Capture of volatile metabolites for tracking the evolution of gibberellic acid in a solid-state culture of Gibberella fujikuroi

The evolution of volatile compounds produced during solid substrate cultivation (SSC) of Gibberella fujikuroi on wheat bran was tracked looking for volatile metabolites related with GA₃ production. Ethyl acetate and isoamyl acetate gave identical profiles and sharp increases that abated as the production of GA₃ began, while ent-kaurene displayed a profile matching that of the development of GA₃. ent-Kaurene is a precursor in the synthesis of gibberellins and was the most abundant compound found.