Distribution of gibberellin biosynthetic genes and gibberellin production in the Gibberella fujikuroi species complex

Gibberella fujikuroi is a species-rich monophyletic complex of at least nine sexually fertile biological species (mating populations, MP-A to MP-I) and more than 30 anamorphs in the genus Fusarium. They produce a variety of secondary metabolites, such as fumonisins, fusaproliferin, moniliformin, beauvericin, fusaric acid, and gibberellins (GAs), a group of plant hormones. In this study, we examined for the first time all nine sexually fertile species (MPs) and additional anamorphs within and outside the G. fujikuroi species complex for the presence of GA biosynthetic genes. So far, the ability to produce GAs was described only for Fusarium fujikuroi (G. fujikuroi MP-C), which contains seven clustered genes in the genome all participating in GA biosynthesis. We show that six other MPs (MPs B, D, E, F, G, and I) and most of the anamorphs within the species complex also contain the entire gene cluster, except for F. verticillioides (MP-A), and F. circinatum (MP-H), containing only parts of it. Despite the presence of the entire gene cluster in most of the species within the G. fujikuroi species complex, expression of GA biosynthetic genes and GA production were detected only in F. fujikuroi (MP-C) and one isolate of F. konzum (MP-I). We used two new molecular marker genes, P450-4 from the GA gene cluster, and cpr, encoding the highly conserved NADPH cytochrome P450 reductase to study phylogenetic relationships within the G. fujikuroi species complex. The molecular phylogenetic studies for both genes have revealed good agreement with phylogenetic trees inferred from other genes. Furthermore, we discuss the role and evolutionary origin of the GA biosynthetic gene cluster.     

Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus niger niaD gene encoding nitrate reductase

Summary A transformation system for Gibberella fujikuroi based on the Aspergillus niger nitrate reductase gene (niaD) was developed. A strain (designated SG140) carrying a non-reverting niaD mutation (niaD11) was generated by screening mutagenised cells for non-growth on nitrate as sole nitrogen source. Transformation frequencies of 1–2 transformants per g DNA were observed when strain SG140 was transformed to nitrate utilisation. Southern blot analyses of niaD⁺ transformants showed that the vector DNA sequences were integrated into the chromosomal DNA. The results demonstrate that the A. niger niaD gene is expressed in G. fujikuroi.     

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.     

Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi

The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.     

Assessment ofGibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.     

调控pH提高分批发酵赤霉素GA3产量

为高效率发酵生产GA₃,对藤仓赤霉菌发酵过程pH进行优化调控研究。采用5L全自动发酵罐,在pH 3.0-5.0条件下,对藤仓赤霉菌菌丝生长及GA₃产量的影响进行了考察,实验数据表明：在pH 4.0条件下,菌比生长速率可获最大值,为0.395/h;而pH 3.0条件下,GA₃比生成速率最大,达到4.43mg/(g?h)。基于不同pH条件下,对菌比生长速率、得率、GA₃比生成速率的影响,提出GA₃分批发酵过程中的pH调控策略,即：0-20h,pH自然;20-50h,pH 4.0;50-80h,pH 3.0-3.5;80h后控制pH为3.5-4.0。在此控制模式下,经过196h发酵GA₃的终产量达到2 224mg/L,GA₃产率44.5mg/g,GA₃生产强度0.242mg/(L?h),分别比不控制pH条件下发酵的数值增长了7.75%、7.74%、8.04%,表明该pH控制策略能增进GA₃发酵生产效率。     

藤仓赤霉菌赤霉素生物合成及代谢调控研究进展

赤霉素是最重要的植物生长调节剂之一,在农业生产中得到越来越广泛的应用,具有广阔的市场前景,但其工业化的高生产成本严重制约着它的广泛应用。近年来,利用生物技术提升赤霉素产量日益成为研究热点。赤霉素生物合成是多种酶协同作用的过程,阐明赤霉素的生物合成机制,利用代谢工程策略调控代谢流量,对提高赤霉素产量至关重要。文中综述了当前藤仓赤霉菌赤霉素生物合成途径、关键酶、环境因素、代谢流调控等方面的研究进展,在代谢调控方面进行了展望,以期为实现赤霉素稳产高产提供思路。     

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.     

Bioprocess strategies and recovery processes in gibberellic acid fermentation

Gibberellic acid (GA₃) is a commercially important plant growth hormone, which is gaining much more attention all over the world due to its effective use in agriculture and brewing industry. Industrially it is produced by submerged fermentation technique using Ascomycetous fungusGibberella fujikuroi. Solid state and immobilized cell fermentation techniques had also been developed as an alternative to obtain higher yield of GA₃. This review summarizes the problems of GA₃ fermentation such as production of co-secondary metabolites along with GA₃, substrate inhibition and degradation of GA₃ to biologically inert compound gibberellenic acid, which limits the yield of GA₃ in the fermentation medium. These problems can be overcome by various bioprocessing strategiese.g. two-stage and fed batch cultivation processes. Further research on bioreactor operation strategies such as continuous and/or extractive fermentation with or without cell recycle/retention system need to be investigated for improvement in yield and productivity. Down stream processing for GA₃ isolation is also a challenge and procedures available for the same have been critically evaluated.     

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.     

Fusarium fractiflexum sp. nov. and two other species within theGibberella fujikuroi species complex recently discovered in Japan that form aerial conidia in false heads

Morphological and molecular phylogenetic analyses were conducted on 12 strains ofFusarium, deposited in MAFF asF. subglutinans (≡F. moniliforme var.subglutinans≡F. sacchari var.subglutinans) orFusarium sp. because they formed aerial conidia in false heads in the dark. These strains were resolved as three distinct species within theGibberella fujikuroi species complex. A new species,F. fractiflexum, and two species new to Japan,F. circinatum andF. concentricum, are described and illustrated and their morphological features are discussed.Fusarium fractiflexum, isolated from diseased yellow leaf spots ofCymbidium spp., is differentiated from other fusaria based on its yellowish colonies and aerial conidia formed in false heads in the dark and in zigzag-like conidial chains under black light. Japanese strains ofF. circinatum also formed elongate, coiled sterile hyphae. Phialidic aerial conidia with a pointed apex and a wedgeshaped base were found inF. concentricum cultured under black light and represent a new diagnostic character of the species, in addition to colonies with alternating concentric rings when cultured on PDA. Based on DNA sequences of the β-tubulin gene and two other loci, strains ofF. fractiflexum were resolved phylogenetically as members of the Asian clade of theG. fujikuroi species complex. In addition, Japanese strains ofF. circinatum andF. concentricum were phylogenetically identical to the ex-type strains.     

Fertility ofFusarium moniliforme from maize and sorghum related to fumonisin production in Italy

Forty-three strains ofFusarium moniliforme isolated from infected maize and sorghum plants in Italy were assayed for their ability to produce fertile crosses with A and F mating population tester strains, in relation to their ability to produce fumonisins on maize substrate. Most of the strains isolated from maize (ear and stalk rot and maize-based feed), producing fumonisin B₁ (FB₁) and B₂ (FB₂) (up to 4,100 and 855 mg/kg, respectively), belonged to the A mating population. All of the strains isolated from sorghum belonged to the F mating population and produced little or no FB₁ and FB₂. This is the first report of the occurrence of mating population F in Europe. Our data on strains from Italy are consistent with previous studies from the United States that found significant differences in sexual fertility and fumonisin production between strains from maize and sorghum.     

Spectrophotometric method for determining gibberellic acid in fermentation broths

A novel method for the quantitative determination of gibberellic acid in fermentation broths has been developed. It is based on the kinetic of the reaction of conversion of gibberellic acid to gibberellenic acid. The method is simple, reliable, faster than most of methods known, and free of the interferences which commonly affect spectrophotometric methods currently in use. Its threshold sensitivity is 0.1 g and its accuracy is greater than 97% for concentrations of gibberellic acid ranging from 0.1 to 1 g l(-1).     

Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases

The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.     

Persistence of [14C] gibberellin A3 and [3H] gibberellin A1 in senescing,ethylene treated Citrus and tomato fruit

The fate of [¹⁴C] gibberellin A₃ and [³H] gibberellin A₁ was examined in senescing fruit of Shamouti orange (Citrus sinensis L. Osbeck) and tomato (Lycopersicon esculentum Mill.). Gibberellin A₃ was highly persistent in Citrus peel (t 1/2=18 days) and to a lesser degree in tomato (t 1/2=5.5 days). Ethylene and ethephon caused a slight enhancement of gibberellin A₃ metabolism in Citrus and tomato fruit, respectively. Gibberellin A₁ was metabolized by Citrus peel at a relatively high rate (t 1/2 < 24 h) and ethylene slightly reduced this rate. It is concluded that the ethylene-induced enhancement of senescence does not involve major effects on the deactivation of applied gibberellins.Abbreviations GA₃ gibberellin A₃ - GA₁ gibberellin A₁     

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.     

The biotransformation of the diterpene 2beta-hydroxy-ent-13-epi-manoyl oxide by Gibberella fujikuroi

Incubation of the diterpene 2beta-hydroxy-ent-13-epi-manoyl oxide with Gibberella fujikuroi afforded in good yield 2beta,6beta-dihydroxy-ent-13-epi-manoyl oxide, 2beta,12beta-dihydroxy-ent-13-epi-manoyl oxide and 2beta,20-dihydroxy-ent-13-epi-manoyl oxide, confirming that although ent-13-epi-manoyl oxide is a final metabolite of a biosynthetic branch in this fungus, more polar derivatives of this compound can be transformed by this micro-organism.     

The microbiological transformation of 7alpha-hydroxy-ent-kaur-16-ene derivatives by Gibberella fujikuroi

The biotransformation of 7alpha-hydroxy-ent-kaur-16-ene (epi-candol A) by the fungus Gibberella fujikuroi gave 7alpha,16alpha,17-trihydroxy-ent-kaur-16-ene and a seco-ring B derivative, fujenoic acid, whilst the incubation of candicandiol (7alpha,18-dihydroxy-ent-kaur-16-ene) and canditriol (7alpha,15alpha,18-trihydroxy-ent-kaur-16-ene) afforded 7alpha,18,19-trihydroxy-ent-kaur-16-ene and 7alpha,11beta,15alpha,18-tetrahydroxy-ent-kaur-16-ene, respectively. The presence of a 7alpha-hydroxyl group in epi-candol A avoids its biotransformation along the biosynthetic pathway of gibberellins, and directs it to the seco-ring B acids route. The 15alpha-hydroxyl group in canditriol inhibits oxidation at C-19 and direct hydroxylation at C-11(beta). The formation of fujenoic acid, from 7alpha-hydroxy-ent-kaur-16-ene, probably occurs via 7alpha-hydroxykaurenoic acid and 7-oxokaurenoic acid, with subsequent hydroxylation at the C-6(beta) position.     

Heterosis and the metabolism of gibberellin A20 in sorghum

The correlation between gibberellin (GA) metabolism and growth rate was investigated using two Sorghum bicolor inbred lines, Hegari and AT×623, and their heterotic F₁ hybrid. Previous studies have demonstrated that this hybrid is taller and has substantially greater shoot dry weights and leaf areas than either parental inbred. [³H]GA₂₀ was applied to the leaf whorl of seedlings and after 24 hours, plants were harvested and separated into roots, shoot cylinders containing the apical meristems, and leaf blades. Chromatographic analyses of metabolites indicated the conversions of [³H]GA₂₀ to [³H]GA_{1,8 and 29}. The conversion of [²H]GA₂₀ to [²H]GA₁ was demonstrated by gas chromatography-selected ion monitoring (GC-SIM). Putative glucosyl conjugates of all of the [³H]GAs were also produced and GA₈ was identified by GC-SIM following enzymic cleavage of the putative [³H]GA₈ glucosyl conjugate fraction. Comparing the genotypes, [³H]GA₂₀ metabolism was more rapid in the shoot cylinders of the hybrid than in the shoot cylinders from inbreds. In the hybrid samples, there was a three-fold increase in the putative conjugate(s) of [³H]GA₁ which was the principal metabolite, and increased production of [³H]GA₈ and the putative conjugates of [³H]GA₂₉ and [³H]GA₈. Conversely, levels of the remaining precursor, [³H]GA₂₀, and its putative conjugate(s) were reduced in the hybrid. The rate of GA₂₀ metabolism was thus positively correlated with growth rate across these sorghum genotypes. This correlation supports a promotive role of GA in the regulation of shoot growth and in the expression of heterosis (hybrid vigor) in sorghum.