Direct biodegradation of eugenol to coniferyl aldehyde and other higher value-added products by Gibberella fujikuroi ZH-34

BACKGROUND: Eugenol is an economically favorable substrate for the microbial biotransformation of aromatic compounds. Coniferyl aldehyde is one kind of aromatic compound that is widely used in condiment and medical industries; it is also an important raw material for producing other valuable products such as vanillin and protocatechuic acid. However, in most eugenol biotransformation processes, only a trace amount of coniferyl aldehyde is detected, thus making these processes economically unattractive. As a result, an investigation of new strains with the capability of producing more coniferyl aldehyde from eugenol is required. RESULTS: We screened a novel strain of Gibberella fujikuroi, labeled as ZH-34, which was capable of transforming eugenol to coniferyl aldehyde. The metabolic pathway was analyzed by high-performance liquid chromatography­mass spectrometry and transformation kinetics. The culture medium and biotransformation conditions were optimized. At a 6 h time interval of eugenol fed-batch strategy, 3.76 ± 0.22 g/L coniferyl aldehyde was obtained, with the corresponding yield of 57.3%. CONCLUSIONS: This work improves the yield of coniferyl aldehyde with a biotechnological approach. Moreover, the fed-batch strategy offers possibility for controlling the target product and accumulating different metabolites

Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi / 中国天然药物

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC values being 35.59 ± 5.37 μmol·L (Caco-2), 24.04 ± 4.70 μmol·L (MCF-7), and 22.24 ± 5.19 μmol·L (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.

Incidence, Molecular Characteristics and Pathogenicity of Gibberella fujikuroi Species Complex Associated with Rice Seeds from Asian Countries

Gibberella fujikuroi species complex (GFSC) was isolated from rice (Oryza sativa L.) seed samples from ten Asian countries and investigated for incidence of GFSC, molecular characteristics, and pathogenicity. Regardless of geographic origin, GFSC was detected with incidences ranging from 3% to 80%. Four species, Fusarium fujikuroi, F. concentricum, F. proliferatum, and F. verticillioides, were found to show an association with rice seeds, with F. fujikuroi being the predominant species. In phylogenetic analyses of DNA sequences, no relationship was found between species, isolates, and geographic sources of samples. Unidentified fragments of the beta-tubulin gene were observed in ten isolates of F. fujikuroi and F. verticillioides. With the exception of three isolates of F. fujikuroi, F. fujikuroi, F. proliferatum, and F. verticillioides were found to have FUM1 (the fumonisin biosynthetic gene); however, FUM1 was not found in isolates of F. concentricum. Results of pathogenicity testing showed that all isolates caused reduced germination of rice seed. In addition, F. fujikuroi and F. concentricum caused typical symptoms of bakanae, leaf elongation and chlorosis, whereas F. proliferatum and F. verticillioides only caused stunting of seedlings. These findings provide insight into the characteristics of GFSC associated with rice seeds and might be helpful in development of strategies for management of bakanae.

Improved calibration of a solid substrate fermentation model

Background: Calibration of dynamic models in biotechnology is challenging. Kinetic models are usually complex and differential equations are highly coupled involving a large number of parameters. In addition, available measurements are scarce and infrequent, and some key variables are often non-measurable. Therefore, effective optimization and statistical analysis methods are crucial to achieve meaningful results. In this research, we apply a metaheuristic scatter search algorithm to calibrate a solid substrate cultivation model. Results: Even though scatter search has shown to be effective for calibrating difficult nonlinear models, we show here that a posteriori analysis can significantly improve the accuracy and reliability of the estimation. Conclusions: Sensibility and correlation analysis helped us detect reliability problems and provided suggestions to improve the design of future experiments.

New stereoisomeric derivatives of jasmonic acid generated by biotransformation with the fungus Gibberella fujikuroi affect the viability of human cancer cells

Background: Several studies have shown that (-)-Jasmonic acid, (+)-7-iso-Jasmonic acid and its methyl ester, methyl jasmonate, have anti-cancer activity in vitro and in vivo, exhibiting selective cytotoxicity towards cancer cells. The degree of activity of these molecules is strongly related to their stereochemistry. The biotransformation of known compounds, natural or synthesized, related to interesting biological activities, generates new molecules displaying new improved properties compared with the original ones, increasing its value and providing new more effective products. Therefore, based on the above rationales and observations, in this work a biotransformation protocol to modify the chemical structure of the plant hormone jasmonic acid by using the fungus Gibberella fujikuroi was established. Results: The three jasmonic acid derivatives obtained, 3(S)-Hydroxy-2(R)-(2Z-pentenyl)-cyclopentane-1(R)-acetic acid (1), 3(R)-Hydroxy-2(R)-(2Z-pentenyl)-cyclopentane-1(R)-acetic acid (2), 3-Hydroxy-2(S)-(2Z-pentenyl)-cyclopentane-1(S)-acetic acid (3), were tested for cell-growth inhibition activity towards the human cancer epithelial cell line, the oral squamous carcinoma cells (KB). The results obtained show that jasmonic acid derivatives (1-3) are active on human cancer cells examined in different concentration ranges, with IC50 value less than of 25 uM. The compound 3, with the same molecular structure of compounds 1 and 2, but with different stereochemistry, was more active confirming that the activity of jasmonate compounds is related to their stereochemistry and to substituents in the cyclopentane ring. In this study, we also tested the potential proapoptotic activity of compound 3, and our data suggest that it, as other jasmonate compounds, is able to trigger apoptotic death in cancer cells. This event may be correlated at an elevation of reactive oxygen species (ROS). Administration of N-acetylcysteine (NAC) prevented compound 3 cytotoxicity...

Producción de ácido giberélico a partir de Gibberella fujikuroi utilizando lodo residual municipal como sustrato / Production of gibberellic acid from Gibberella fujikuroi using municipal sewage sludge as a substrate / Produção de ácido giberélico desde Gibberella fujikuroi usando lodo residual municipal como substrato

Objetivo. Utilizar lodos residuales municipales (LRM) provenientes de una planta de tratamiento de aguas residuales ubicada en Toluca, Estado de México, para cultivar al hongo Gibberella fujikuroi en fermentación sumergida y producir ácido giberélico (AG3). Materiales y métodos. Se utilizó Gibberella fujikuroi (CDBB:268). Para la obtención de AG3 se verificó la producción empleando como sustrato al medio de cultivo estándar (MCE). La determinación de (AG ) fue por cromatografía líquida de alta resolución (HPLC) con un equipo Varian, 9050,9012. Se obtuvieron 6 muestras de lodos de una planta tratadora de aguas residuales en Toluca, Estado de México y fueron caracterizados. Finalmente ambos sustratos (LRM y MCE) se usaron en fermentación sumergida y por extracción se obtuvo el AG3 cuantificado por HPLC. Resultados. La caracterización del LRM demostró que el contenido de materia orgánica (MO) es de 5.20 % (m/v) y nitrógeno total (N T) de 0.25 % (m/v), dicha composición está dentro del rango como sustrato para la producción del AG3 por Gibberella fujikuroi. El hongo se cultivó durante 3, 8, 13 y 30 días utilizando lodo residual estéril con una humedad del 95.6 %(m/v) y en medio de cultivo estándar (MCE), las muestras se procesaron y analizaron por cromatografía líquida de alta resolución (HPLC), donde la producción de AG3 en el LRM fue de 460.06 mg/L para 30 días en fermentación sumergida a un pH de 4.0 y 1014.46 mg/L para el control. Conclusiones. El contenido nutritivo del LRM es adecuado para el crecimiento del hongo Gibberella fujikuroi y la producción de AG3 empleando como sustrato LRM.

Objective. To use municipal sewage sludge (LRM) from a wastewater treatment plant located in Toluca, State of Mexico, to grow the fungus Gibberella fujikuroi in submerged fermentation and to produce gibberellic acid (AG3). Materials and methods. We used Gibberella fujikuroi (CDBB: 268). To obtain AG3, production was verified using as a substrate the standard culture medium (MCE). Gibberellic acid determination was done with high performance liquid chromatography (HPLC) with a Varian 9050.9012 equipment. We obtained 6 samples of sludge from a wastewater treatment plant in Toluca, State of Mexico, that were then characterized. Finally, both substrates (LRM and MCE) were used in submerged fermentation, and GA3 was obtained by extraction and quantified using HPLC. Results. The LRM characterization showed that the organic matter content (MO) is of 5.20% (w/v) and the total nitrogen content (N T) is of 0.25% (w/v). Such composition is within the range as a substrate for the production of AG3 by Gibberella fujikuroi. The fungus was cultivated for 3, 8, 13 and 30 days in sterile sewage sludge with a moisture content of 95.6% (w/v) and in standard culture medium (MCE). Samples were processed and analyzed by high performance liquid chromatography (HPLC). Production of AG3 in the LRM was of 460.06 mg/L after 30 days in submerged fermentation at pH 4.0, and of 1,014.46 mg/L in the control. Conclusion. The nutrient content of LRM is suitable for the growth of the fungus Gibberella fujikuroi and for the production of GA3 when used as a substrate.

Objetivo. Usar lodos residuais municipais (LRM) procedentes de uma estação de tratamento de águas residuais de Toluca, Estado de México, para cultivar o fungo Gibberella fujikuroi em fermentação submersa e produzir ácido giberélico (AG3). Materiais e métodos. Foi utilizado Gibberella fujikuroi (CDBB: 268). Para obter AG3 foi verificada a produção empregando como substrato o meio de cultura padrão (MCE). A determinação de ácido giberélico foi por cromatografia líquida de alta eficiência (HPLC) com um equipo Varian, 9050,9012. Foram obtidas seis amostras de lodos de uma estação de tratamento de águas residuais em Toluca, Estado de México e foram caracterizadas. Finalmente, ambos os substratos (LRM e MCE) foram utilizados em fermentação submersa e por extração foi obtido o AG3 quantificado por HPLC. Resultados. A caracterização do LRM demonstrou que o teor de matéria orgânica (MO) é 5,20% (m/v) e o nitrogênio total (N T) 0,25% (m/v), esta composição está dentro do intervalo como substrato para a produção de AG3 por Gibberella fujikuroi. O fungo foi cultivado por 3, 8, 13 e 30 dias usando lodo residual estéril com um teor de umidade de 95,6% (m/v) e em meio de cultura padrão (MEC), as amostras foram processadas e analisadas por cromatografia líquida de alta resolução (HPLC), onde a produção de AG3 no LRM foi 460,06 mg/L para 30 dias em fermentação submersa em pH 4,0 e 1.014,46 mg/L para o controle. Conclusões. O teor nutritivo do LRM é adequado para o crescimento do fungo Gibberella fujikuroi e a produção de AG3, empregando como substrato o LRM.

Mating Types and Optimum Culture Conditions for Sexual State Formation of Fusarium fujikuroi Isolates

Twenty-five isolates of Fusarium fujikuroi acquired from rice seeds and rice plants evidencing symptoms of Bakanae disease were evaluated to determine their mating types and characterize the formation of their sexual state. The mating types of the isolates were evaluated via multiplex PCR with the diagnostic primers of the mating-type (MAT) region: GFmat1a, GFmat1b, GFmat2c, and GFmat2d. Among the 25 isolates, 11 were identified as MAT-1 (male), and 14 as MAT-2 (female). Four MAT-1 isolates and three MAT-2 isolates were mated and cultured to evaluate the optimal culture conditions for the production of their sexual states. Among four tested media, 10% V8 juice agar proved optimal for the perithecial production of the isolates. The isolates also generated the largest numbers of perithecia when incubated at 23degrees C in alternating cycles of 12 hr fluorescent light and NUV fluorescent light and 12 hr darkness.

Genotypic Identification of Fusarium subglutinans, F. proliferatum and F. verticillioides Strains Isolated from Maize in Austria

Gibberella fujikuroi is species complex. This species complex includes Fusarium tabacinum, F. moniliforme (= F. verticillioides), F. nygamai, F. proliferatum as well as F. subglutinans. Our objective was to develop a technique to differentiate between isolates of F. subglutinans, F. proliferatum and F. verticillioides. Thirty-two strains of F. subglutinans, six strains from F. verticillioides and five strains of F. Proliferatum isolated from maize in Austria were studied using random amplified polymorphic DNA (RAPD). F. subglutinans strains clustered very closely, with similarity ranging from 87~100%. On the other hand, all the amplification patterns of F. verticillioides were identical, as well as in the case of F. proliferatum. Our results indicated that these Fusaria species are distinct species and hence RAPD markers can be quick and reliable for differentiating them.