Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.     

Influence of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> mycelia culture conditions on antimicrobial metabolite production

Enokipodins A, B, C, and D are α-cuparene-type sesquiterpenoids antimicrobial metabolites produced in the stationary stage of Flammulina velutipes mycelia development in malt extract broth. This study assessed the influence of nutritional and environmental factors on F. velutipes mycelia culture for the production of these metabolites. The mycelia growth and antimicrobial activity were assessed by determining dry matter and the diffusion in agar method, respectively. The best F. velutipes mycelia growth was observed in dextrose potato broth, and greater antimicrobial metabolite production occurred in complete Pontecorvo’s culture medium. Environmental modifications, such as a rise in temperature from 25° to 37°C on the 15th day of F. velutipes mycelia culture in malt extract and peptone broth, also optimized antimicrobial metabolite production. The metabolites produced in these treatments were correlated with the enokipodins A and B in thin-layer chromatography (TLC) and the antifungal activity test by TLC bioautography. This study showed that there was no correlation between biomass production and antimicrobial metabolite production, but there may be a correlation between culture medium composition and enokipodins biosynthesis.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Afritoxinones A and B,dihydrofuropyran-2-ones produced by Diplodia africana the causal agent of branch dieback on Juniperus phoenicea

Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Phoenicean juniper in Italy. Additionally, six others known metabolites were isolated and characterized: oxysporone, sphaeropsidin A, epi-sphaeropsidone, R-(−)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS¹,6R¹,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR¹,6R¹,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host (Phoenicean juniper) and non-host plant (holm oak, cork oak and tomato) by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. This study represents the first report of secondary metabolites produced by D. africana. In addition, the taxonomic implications of secondary metabolites in Botryosphaeriaceae family studies are discussed.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

海洋真菌Arthrinium sp. UJNMF0008代谢产物的研究

为丰富海洋真菌的化学多样性,发现海洋真菌活性代谢产物,对海洋沉积物来源真菌Arthriniumsp.UJNMF0008的化学成分及其生物活性进行研究,采用硅胶柱色谱、凝胶柱色谱、反向柱色谱和高效液相色谱等方法从海洋沉积物来源真菌Arthriniumsp.UJNMF0008的发酵提取物中分离到5个化合物,通过核磁共振、质谱等方法,结合文献对照,鉴定了化合物的结构分别为arthoneF(1)、arthoneG(2)、sydoxanthoneC(3)、(3R,4R)-cis-4-hydroxymellein(4)和2-(2′S-hydroxypropyl)-5-methyl-7-hydroxychromone(5),其中化合物1和2是新化合物,化合物3首次从该属真菌中分离到。活性测试显示,化合物1~5在50μmoL/L的测试浓度下均没有表现出明显的抗氧自由基活性、抗菌活性以及NO释放抑制活性。     

Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

Patterns of protein expression upon adding sugar and elicitor to the cell culture of Eschscholtzia californica

The optimal timing of elicitation was determined for the production of benzophenanthridine alkaloids (BPAs) by Eschscholtzia californica cell culture. Upon elicitation, 7-day old cells produced more alkaloids than 14-day old cells (5.1 times for sanguinarine and 2.7 times for dihydrosanguinarine). We presumed that these alkaloids are growth-rate-associated secondary metabolites in E. californica cell culture. Although the specific productivity of alkaloids were higher in 7-day old culture, the total cell mass of 7-day old culture was about half that of 14-day old culture. In order to increase the overall productivity, sucrose was added to the 14-day old culture before the addition of elicitor. By this way, cells in the stationary phase (14-day old culture) could be switched to the cells in the logarithmic growth phase (similar to 7-day old culture). Total production of alkaloids was increased by adding sucrose; especially the production of sanguinarine was increased as high as 5.7 times of the control. To find out the protein level changed by the elicitation, proteins extracted from whole cell were separated by using two-dimensional gel electrophoresis. The patterns of the gels were different and little correlation among the proteins could be observed. And Western blotting was employed to check the expression level of selected five enzymes, these enzymes believed to be involved in BPAs production, resulting in up-regulated with elicitor addition.     

Stationary and microcarrier cell culture processes for propagating Japanese encephalitis virus

Inactivated mouse-brain-derived vaccines for Japanese encephalitis virus (JEV) have been used for many years. Recently, attempts have been made to employ cultured Vero cells to replace mouse brain tissues for developing cell-culture-derived vaccines that will be more suitable for worldwide usage. In this study, JEV replication processes in Vero and BHK cells and between stationary and microcarrier culture systems were investigated. Our results demonstrated that a stationary Vero cell culture system produced higher viral titers of JEV, including the Beijin-1 vaccine strain and the attenuated strain CH2195LA, than microcarrier culture did. BHK cells showed less significant differences in their replication kinetics between stationary and microcarrier cultures. Reducing serum concentration during infection led to an overall decrease of JEV production in Vero cells but an increase in BHK cells. By establishing a complete serum-free Vero cell culture, the microcarrier system resulted in a more than 4-log lowered yield compared to that of the stationary culture for JEV production. Thus, the stationary culture is the most efficient system for JEV production from cultured Vero cells.     

Phytotoxins produced byTubakia dryina

Tubakia dryina, the causal agent of red oak (Quercus rubra) leaf spot, produced the phytotoxins isosclerone, 3-hydroxyisosclerone, 6-hydroxyisosclerone and 6-hydroxymellein in liquid culture. All toxins caused large necrosis when placed on leaves of red oak. Necrosis was also caused on white oak and eight different weed species. All toxins were non-specific phytotoxins.     

Isolation of secondary metabolites of Aspergillus ochraceus by HPLC

A method is described for the isolation and purification of ochratoxin A, ochratoxin B, ochratoxin ß mellein, 4-hydroxymellein and penicillic acid produced byAspergillus ochraceus in a synthetic liquid medium. Ochratoxin α, which was not found in the culture medium, was obtained by acid hydrolysis of ochratoxin A. A high pressure liquid Chromatograph equipped with Lichrosorb 100 and Lichrosorb RP-18 columns and UV and/or Refractive Index detectors was used.     

Effect of Metabolites Produced by Trichoderma harzianum Biotypes and Agaricus bisporus on Their Respective Growth Radii in Culture

Trichoderma harzianum biotypes Th1, Th2, and Th3 produced volatile metabolites in vitro which had similar fungistatic effects on the growth of Agaricus bisporus. Metabolites present in agar colonized by these strains also inhibited mycelial growth of A. bisporus, although the reduction in growth was less in the presence of metabolites produced by biotype Th2 than that in the presence of metabolites produced by Th1 or Th3. A. bisporus produced metabolites in liquid culture that inhibited the growth of Th1 and Th3 but stimulated the growth of Th2. A compound(s) responsible for the inhibition and stimulation was extracted from A. bisporus culture filtrate and from compost-grown fruit bodies with n-butanol, but the identity of the compound(s) was not determined. We suggest that the stimulation of Th2 by metabolites produced by A. bisporus and the relatively low level of inhibition of A. bisporus by Th2 facilitate colonization of compost by both fungi. However, as compost colonization reaches a maximum, a change in the competitive balance in favor of Th2 results in the inhibition of fruit body production by A. bisporus and the devastating green mold epidemics affecting mushroom production.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.)

Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, was investigated to establish the effect on plant growth of growth substances produced by the bacteria. Thin-layer chromatography, high-pressure liquid chromatography, and bioassay were used to separate and identify plant growth substances produced by the bacteria in liquid culture. Indole acetic acid and indole lactic acid were produced by A. brasilense from tryptophan. Indole acetic acid production increased with increasing tryptophan concentration from 1 to 100 μg/ml. Indole acetic acid concentration also increased with the age of the culture until bacteria reached the stationary phase. Shaking favored the production of indole acetic acid, especially in a medium containing nitrogen. A small but biologically significant amount of gibberellin was detected in the culture medium. Also at least three cytokinin-like substances, equivalent to about 0.001 μg of kinetin per ml, were present. The morphology of pearl millet roots changed when plants in solution culture were inoculated. The number of lateral roots was increased, and all lateral roots were densely covered with root hairs. Experiments with pure plant hormones showed that gibberellin causes increased production of lateral roots. Cytokinin stimulated root hair formation, but reduced lateral root production and elongation of the main root. Combinations of indole acetic acid, gibberellin, and kinetin produced changes in root morphology of pearl millet similar to those produced by inoculation with A. brasilense.     

Differential Sensitivity of Wheat Embryos against Extracts Containing Toxins of Septoria nodorum: First Steps towards in vitro Selection

As a first step towards the development of an in vitro -selection system for septoria nodorum blotch resistance, wheat embryo culture on media containing extracts from Septoria nodorum was established. Extracts prepared from inoculated wheat grains had a toxic activity. Control extracts from uninoculated grains showed at least a 10-fold lower toxic activity. Two wheat breeding lines susceptible to Septoria nodorum showed reduced growth in the presence of the fungal extract whencompared to a breeding line known to have good resistance in the field. A test with seven additional wheat lines showed a good agreement between field resistance of the ear and embryo resistance. Mellein is one of the toxins produced by Septoria nodorum and was used in pure form for in vitro -selection. It showed toxic effects at 50 μg/ml, a concentration which is about 200-fold higher than the mellein concentration in the diluted extract with embryotoxic activity. This indicates the importance of additional toxic compounds in the crude extract. Mellein acted non-selectively on embryos of the different cultivars.     

Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts

Synthetic activity of 6-hydroxymellein, the immediate precursor of carrot phytoalexin 6-methoxymellein, from acetyl-CoA and malonyl-CoA was induced in carrot cell extracts when the root disks were treated with CuCl2 or oligogalacturonide elicitor. These elicitors showed specific inducing activity of phytoalexin production and did not affect fatty acid synthesis in carrot tissues which may share some common properties with 6-hydroxymellein biosynthesis. 6-Hydroxymellein production was an NADPH-dependent process and, in the absence of the reagent, triacetic acid lactone was produced as a derailment product of the reaction process. This finding suggested that the reduction of the double bond at the 3,4-position of the phytoalexin takes place during the elongation of the poly(oxomethylene) chain. This NADPH-dependent reduction seems to occur at the triacetate stage before the condensation of the third malonyl-CoA as the conversion of carbonyl to hydroxyl group.     

Replicative aging of the yeast does not require DNA replication

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.     

Secondary metabolites ofPenicillium bilaii strain PB-50

A phosphate-solubilizing strain ofPenicillium bilaii was tested for the production of gliotoxin and other toxic compounds. The strain was fermented under five different conditions to allow the expression of various metabolites, including gliotoxin. These included Czapek-yeast extract medium under both shaken and still conditions as well as Czapek-yeast extract/malt extract/peptone medium and sucrose/glycerol medium in shake flasks. In addition, culture filtrate from an industrial fermentation of the fungus was examined. No gliotoxin was produced in any of the media. No other expectedP. bilaii metabolites were found. Three compounds were identified in all samples: dibutyl phthalate, 1-(4-hydroxy-phenyl)ethanone and 4-hydroxy-3,6-dimethyl-2H-pyran-2-one. The production of other metabolites was dependent on the culture conditions. Two hyalodendrin derivatives were found in some fermentations and two related compounds were tentatively identified. None of the compounds found have been reported as toxic. The identity of the culture was confirmed by comparison with the ex-type culture ofP. bilaii.