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.     

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.     

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.     

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.     

Carotenoid synthesis in Streptomyces setonii ISP5395 is induced by the gene crtS,whose product is similar to a sigma factor

In many species of actinomycetes, carotenogenesis can be photoinduced. The capacity to respond to photoinduction is, however unstable and, in various strains of Streptomyces, is lost at a relatively high frequency. In Streptomyces setonii ISP5395, which normally produces no carotenoids, carotenoid-producing mutants can be obtained following protoplast regeneration. We report here the characterization of a gene, crtS, which was isolated from one such mutant and can confer on wild-type S. setonii ISP5395 cells the capacity to synthesize carotenoids. Sequence analysis of crtS reveals an open reading frame, which shows homology to genes that encode alternative sigma factors in Bacillus subtilis. We propose that crtS encodes a sigma factor which is necessary for the expression of a cryptic gene(s) for carotenoid biosynthesis in S. setonii ISP5395.     

Homologous recombination and allele replacement in transformants of Fusarium fujikuroi

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others. Received: 5 November 1999 / Accepted: 16 March 2000     

Isolation and Characterization of the Gibberellin Biosynthetic Gene Cluster in Sphaceloma manihoticola

Gibberellins (GAs) are tetracyclic diterpenoid phytohormones that were first identified as secondary metabolites of the fungus Fusarium fujikuroi (teleomorph, Gibberella fujikuroi). GAs were also found in the cassava pathogen Sphaceloma manihoticola, but the spectrum of GAs differed from that in F. fujikuroi. In contrast to F. fujikuroi, the GA biosynthetic pathway has not been studied in detail in S. manihoticola, and none of the GA biosynthetic genes have been cloned from the species. Here, we present the identification of the GA biosynthetic gene cluster from S. manihoticola consisting of five genes encoding a bifunctional ent-copalyl/ent-kaurene synthase (CPS/KS), a pathway-specific geranylgeranyl diphosphate synthase (GGS2), and three cytochrome P450 monooxygenases. The functions of all of the genes were analyzed either by a gene replacement approach or by complementing the corresponding F. fujikuroi mutants. The cluster organization and gene functions are similar to those in F. fujikuroi. However, the two border genes in the Fusarium cluster encoding the GA₄ desaturase (DES) and the 13-hydroxylase (P450-3) are absent in the S. manihoticola GA gene cluster, consistent with the spectrum of GAs produced by this fungus. The close similarity between the two GA gene clusters, the identical gene functions, and the conserved intron positions suggest a common evolutionary origin despite the distant relatedness of the two fungi.     

Complementation between mutants of Phycomyces deficient with respect to carotenogenesis

Summary White and red mutants of Phycomyces, derived from two independent wild types (yellow) by mutagenesis using nitrosoguanidine, either in a single step (26 white, 5 red mutants), or in two steps (10 white mutants, from one of the red mutants) were studied with respect to complementation in heterokaryons. The tests clearly establish the involvement of three and only three genes, here named carA, carB, and carB. The carA and the carR mutants are white, the carA mutants do not accumulate phytoene, the carB mutants do. The carR mutants are red and accumulate lycopene. The two step mutants are either carA and carR, or carB and carR double mutants. A few of the white mutants obtained in a single mutagenization step are affected in carA and carR. They may be polar mutants in an operon or accidental double mutants.     

Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus

Summary Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light. We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants. A carR mutant produces the same carotenoids in the dark as the wild type grown in the light. This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system. A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA. In the dark, the carA mutant produces high levels of phytoene, the first C₄₀ colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type. Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene. This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others. Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids. The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect.     

In vitro carotenogenesis by wild type and mutants of Phycomyces blakesleeanus

Cell extracts from shake cultures of the wild type and six mutant strains of Phycomyces converted [2-¹⁴C] MVA into carotenes, squalene and prenyl phosphates. Oxygen was required for the desaturation of phytoene. When compared with the wild type, cells extracts of carB and carR mutants are much less effective in phytoene dehydrogenation and lycopene cyclization, respectively. This confirms previous conclusions about the biochemical functions of the carB and carR genes, which were based on genetic and in vivo studies. CarA strain mutants accumulate, in vivo, much less β-carotene than the wild type. This correlates with a 10-fold decrease in carotenogenesis in vitro. The addition of retinol to incubations of cell extracts of the wild type and C2 strains stimulated β-carotene formation. Both carB and carR mutants show enhanced total carotenogenic activities in vitro and the carS mutant shows a higher β-carotene-synthesizing activity than the wild type. It is suggested that the feed-back regulatory mechanism known to control this pathway operates at the level of enzyme synthesis.     

Loss of Gibberellin Production in Fusarium verticillioides (Gibberella fujikuroi MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster

Fusarium verticillioides (Gibberella fujikuroi mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the Gibberella fujikuroi species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to Fusarium fujikuroi (G. fujikuroi MP-C) and Fusarium konzum (MP-I), although most members of the G. fujikuroi species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in F. verticillioides (MP-A) is due to the loss of a majority of the GA gene cluster as found in F. fujikuroi. The remaining part of the cluster consists of the full-length F. verticillioides des gene (Fvdes), encoding the GA₄ desaturase, and the coding region of FvP450-4, encoding the ent-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of F. fujikuroi. The GA production capacity of F. verticillioides was restored by transforming a cosmid with the entire GA gene cluster from F. fujikuroi, indicating the existence of an active regulation system in F. verticillioides. Furthermore, the GA₄ desaturase gene des from F. verticillioides encodes an active enzyme which was able to restore the GA production in a corresponding des deletion mutant of F. fujikuroi.     

A study of the nitrate and nitrite discharge from the mutant cells of <Emphasis Type="Italic">Neurospora crassa</Emphasis> lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.