Thesis (Ph. D.)--University of Wisconsin--Madison, 1946. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [65]-69).

Thesis (Ph.D.) - University of St Andrews, March 2009. / Restricted until 17th March 2010.

University, Diss., 2003--Marburg.

No description available.

No description available.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 203-220).

Univ., Diss.--München, 2000.

Tesis presentada a la Universidad de Chile para optar al grado académico de Magister en Bioquímica área de especialización en Bioquímica Ambiental y Memoria para optar al título profesional de Bioquímico / Memoria de título de bioquímico / No autorizada por el autor para ser publicada a texto completo / El hongo filamentoso Fusarium fujikuroi produce diversos metabolitos secundarios entre los que se encuentran las giberelinas (GAs), diterpenoides tetracíclicos derivados del ácido mevalónico de interés agronómico debido al efecto regulador que presentan sobre el crecimiento y desarrollo de las plantas. El interés comercial de estas moléculas y la alta eficiencia del sistema fúngico han llevado a caracterizar su biosíntesis a nivel de las reacciones químicas, las enzimas y los genes implicados en el proceso. La secuencia biosintética consiste principalmente en reacciones de oxidación, las que son catalizadas por monooxigenasas (MO) P450 codificadas por genes agrupados en un cluster. En este hongo la biosíntesis de GAs se induce en condiciones de carencia de compuestos nitrogenados y es inhibida por amonio o glutamina debido a la represión de los genes. Este mecanismo de regulación está mediado principalmente por el regulador global AREA, un factor de transcripción cuya actividad depende de los niveles de nitrógeno, aunque también podrían participar otros elementos regulatorios específicos para esta vía metabólica. Con el objeto de contribuir a la comprensión de los mecanismos de regulación de la biosíntesis de las GAs fúngicas, en este trabajo de Tesis se investigó si los genes de GAs de F. fujikuroi se expresan como proteínas activas en tres especies de hongos filogenéticamente relacionadas a F. fujikuroi: Fusarium oxysporum, Fusarium graminearum y Aspergillus nidulans. Estos hongos no poseen los genes de la biosíntesis de GAs pero contienen el regulador global AREA. Interesó determinar si la complementación con el cluster de genes de F. fujikuroi es suficiente para generar la capacidad de biosintetizar GAs en estas especies y si las transformantes presentan el mecanismo de represión por amonio. Se caracterizó la producción de GAs en cultivos líquidos mediante cromatografía de gases acoplada a espectrometría de masas (GC-MS) y las actividades de las distintas oxidasas de GAs se ensayaron mediante sustratos marcados con [14C] e identificando los productos por cromatografía en capa fina y cromatografía líquida de alta resolución. Se encontró que las transformantes T6, T11 y T1 de F. oxysporum así como la transformante T2 de F. graminearum presentan la capacidad de biosintetizar GAs en un medio líquido carente de compuestos nitrogenados. Principalmente sintetizan GAs 3β-hidroxiladas y 19-γ10-lactónicas (19C) como GA4, GA7 y GA3, además algunas GAs no hidroxiladas (GA9, GA24, GA25) y entkaurenoides, que son productos laterales de la vía biosintética. Este patrón de productos corresponde al que presenta la cepa silvestre IMI28589 de F. fujikuroi, sistema fúngico que sintetiza GA3 como producto final a partir de intermediarios 3β-hidroxilados. Estos resultados indican que los genes de la biosíntesis de GAs de F. fujikuroi se expresan y generan enzimas activas tanto en F. oxysporum como en F. graminearum. En particular, la metabolización del ácido ent-[14C]kaurenoico hasta [14C]GA14 y posteriormente hasta [14C]GA3 demostró que la MO P450-1 (GA14 sintasa) presenta las actividades de 7-oxidasa y de 3β-hidroxilasa, como en F. fujikuroi. Por otra parte, la MO P450-2 (GA20 oxidasa) forma el producto lactónico [14C]GA9 y su derivado [14C]GA40, a partir del sustrato [14C]GA12. En estos ensayos también se detectaron las actividades de 13-hidroxilasa y de desaturasa aunque con diferentes proporciones en las distintas transformantes, en concordancia con el análisis de GAs endógenas. El efecto represor del nitrato de amonio se determinó a través de la actividad de la GA20 oxidasa en la transformante de F. graminearum T2, donde se encontró que la velocidad de utilización de [14C]GA12 fue 5 veces menor en un medio con nitrato de amonio 4,8 g/L que en un medio sin amonio, en forma similar a la cepa silvestre IMI28589 de F. fujikuroi (siete veces menor en presencia de amonio). Esto sugiere que estaría operativo en F. graminearum el mecanismo de represión por compuestos nitrogenados. Para la transformante T6 de F.oxysporum el resultado fue menos claro, ya que se obtuvieron productos en presencia de amonio que no corresponden a la lactona [14C]GA9 o a su derivado [14C]GA40 (los principales productos de la GA20 oxidasa). La identificación de los productos por GC-MS permitirá confirmar si corresponden a la actividad de oxidasas inespecíficas y si la represión por nitrato de amonio está presente en esta especie. A diferencia de las transformantes de F. oxysporum y de F. graminearum, en cultivos de la transformante de A. nidulans no se detectó actividad de ninguna de las oxidasas de GAs en los ensayos con los sustratos marcados con [14C]. Esto sugiere que los genes de F. fujikuroi no se expresarían en A. nidulans, una especie filogenéticamente más alejada de F. fujikuroi que F. oxysporum y F. graminearum, a pesar de que AREA está presente en esta especie. En conclusión, los resultados obtenidos sugieren que las dos especies de Fusarium investigadas, F. oxysporum y F. graminearum, contienen los elementos regulatorios (AREA y/u otros) requeridos para la expresión de los genes de la biosíntesis de GAs en condiciones de carencia de nitrógeno. Estos factores no serían específicos para esta vía, ya que se encontraron en dos especies fúngicas que no contienen genes de la biosíntesis de GAs y no sintetizan estos diterpenos. / The filamentous fungus Fusarium fujikuroi synthesizes several secondary metabolites including gibberellins (GAs), tetracyclic diterpenoids derived from mevalonic acid that have an agronomic interest since they are plant growth regulators. Because of the high efficiency of the fungal system and its commercial interest GA biosynthesis has been characterized at the level of chemical reactions, enzymes and genes. The fungal GA biosynthetic pathway is based on oxidative reactions catalyzed by P450 monooxygenases (MO) codified by a gene cluster. GA biosynthesis is induced in the absence of nitrogenated compounds and inhibited by ammonia or glutamine due to repression of gene expression. The global regulator AREA, a transcription factor dependent on nitrogen levels, mediates this effect, together with other possible specific regulatory elements. In order to contribute to the understanding of GA biosynthesis regulation in F. fujikuroi in this work we investigated if the F. fujikuroi GA biosynthesis genes are expressed as active enzymes in three fungal species phylogenetically related to F. fujikuroi: Fusarium oxysporum, Fusarium graminearum and Aspergillus nidulans. These species do not contain the GA biosynthesis genes but contain the global regulator AREA. It was investigated if complementation with the F. fujikuroi gene cluster is enough to restore GA biosynthesis in these fungal species and if the transformants present ammonium repression of the GA genes as in F. fujikuroi. GA biosynthesis was determined in liquid cultures by gas chromatography coupled to mass spectrometry (GC-MS) and the activities of GA oxidases were assayed with [14C]-labelled substrates by identifying the respective products by thin layer chromatography or high performance liquid chromatography. F. oxysporum transformants T6, T11 and T1 as well as F. graminearum T2 synthesized GAs in liquid cultures that do not contain nitrogenated compounds. These were mainly 19-γ10-lactonic, 3β-hydroxylated GAs (C19; GA4, GA7 and GA3) together with non-hydroxylated GAs (GA9, GA24,GA25) and ent-kaurenoids, lateral products of the GA biosynthetic pathway. This product pattern corresponds to that of the wild-type F. fujikuroi strain IMI28589 that synthesizes GA3 as final product from 3β-hydroxylated intermediates. These results demonstrate that the F. fujikuroi GA biosynthesis genes are expressed as active enzymes in F. oxysporum as well as in F. graminearum. Particularly the conversion of ent-[14C]kaurenoic acid into [14C]GA14 and further into [14C]GA3 demonstrated that in F. oxysporum and F. graminearum GA14 synthase presents 7- oxidase and 3β-hydroxylase activities as in F. fujikuroi. On the other hand, in these transformants GA20 oxidase gave the 19-γ10-lactonic product [14C]GA9 or its derivative [14C]GA40 from [14C]GA12. 13-hydroxylase and desaturase activities were also detected in these assays even when in different proportions in the distinct transformants in agreement with endogenous GA analysis. Ammonium nitrate repressor effect was investigated over GA20 oxidase that was assayed in F. graminearum T2 liquid cultures. The rate of [14C]GA12 conversion was found to be 5 times less in 4,8 g/L ammonium-containing media than in the absence of ammonium, similar to that found for F. fujikuroi IMI28589 cultures (7 times less [14C]GA12 conversion in media containing ammonia). This suggests that the mechanism of repression by nitrogenated compounds would be active in F. graminearum. For F. oxysporum T6 a less clear result was obtained since the products formed in the presence of 4,8 g/L ammonium nitrate do not include [14C]GA9 or [14C]GA40, the main products of GA20 oxidase. GC-MS identification of these products will allow to confirm if they correspond to unspecific oxidation products and if ammonium repression is also present in F. oxysporum. In contrast to F. oxysporum and F. graminearum, the cultures of A. nidulans complemented with the F. fujikuroi GA biosynthesis genes did not present activity of any of the GA oxidases in assays with ent-[14C]kaurenoic acid, [14C]GA12 or [14C]GA4. This suggests that the F. fujikuroi genes would not be expressed in A.nidulans, a species phylogenetically less related to F. fujikuroi, even when it contains AREA. Altogether, the results obtained suggest that the two Fusarium species investigated contain the regulatory elements required for the expression of the GA biosynthesis genes (AREA and/or others) in the absence of nitrogenated compounds. These factors would not be specific for the GA pathway since they are present in two fungal species that do not contain the GA biosynthesis genes and do not synthesize GAs. / Fondecyt

Orientador: Sandra Regina Pombeiro Sponchiado / Banca: Maria Lucia Gonsales da Costa Araújo / Banca: Suraia Said / Resumo: Devido ao crescente interesse na produção de substâncias ativas a partir de fontes naturais, os fungos vêm sendo considerados um grupo importante de microrganismos em função do seu enorme potencial de exploração, representando uma fonte inesgotável para a obtenção de vários produtos biotecnológicos industriais. Dentre estes produtos, o pigmento melanina produzido pelo fungo Aspergillus nidulans tem considerável potencial biotecnológico para ser usado em formulações cosméticas, principalmente pelas suas atividades antioxidante, antiinflamatória e fotoprotetora. No entanto, para uma possível aplicação prática desta substância é necessário estabelecer as condições ótimas de cultivo do fungo para a produção em larga escala com um custo menor comparado à melanina sintética. Neste contexto, o objetivo deste trabalho foi avaliar a influência do volume de meio de cultura, do tempo de incubação e da utilização de resíduos industriais (água de maceração de milho, bagaço de cana de açúcar e melaço) como fontes de nutrientes no crescimento e produção de pigmento pelas linhagens MEL1 e MEL2 de Aspergillus nidulans. Os resultados mostraram que na presença de 2% de água de maceração de milho o crescimento celular foi maior enquanto que na concentração de 0,2% houve um aumento na produção de pigmento pelas linhagens em estudo. Quando melaço (1% e 10%) foi usado como fonte de carbono, a produção de pigmento foi inibida em ambas as concentrações, enquanto um alto rendimento de biomassa foi obtido com 10% de melaço. A suplementação do meio com hidrolisado do bagaço da cana-de-açúcar não teve um efeito positivo na produção de pigmento, embora tenha proporcionado um aumento no crescimento das linhagens. Também foi observado que a morfologia do fungo (tamanho do "pellet") influencia a produção de pigmento, sendo que na cultura... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to an increasing interest in the production of actives substances from natural sources, fungi has been considered an important group of microorganisms in function of its enormous potential for exploitation, representing an inexhaustible source to obtain several industrials biotechnology products. Among these products, the melanin pigment produced by fungi Aspergillus nidulans has considerable biotechnological potential to be used in cosmetic formulations, mainly for its antioxidant, anti-inflammatory and photoprotetor activities. However, for a possible practical application of this substance, is necessary to establish the optimal conditions of the fungal culture for large scale production with low cost compared to synthetic melanin. In this context, the objective of this work was to evaluate the influence of volume of culture medium, period of incubation and the use of by-products industrial (corn steep liquor, sugar cane bagasse and molasses) as nutritional source on growth and pigment production by strains MEL 1 and MEL 2 from Aspergillus nidulans. The results showed that, in the presence of 2% corn steep liquor, the cell growth was high while at concentration of 0,2% occurred an increase in the pigment production. When molasses (1 or 10%) was used as carbon sources, the pigment production was inhibited in both concentrations while a high yield of biomass was obtained with 10% molasses. The supplementation of medium with sugar cane bagasse hydrolysate didn't has a positive effect on pigment production but it provided an increase in the fungal growth. Also was observed that the fungal morphology (pellet size) influenced the production of pigment, being the smaller "pellets" more suitable for maximum production of the pigment. Thus, the results obtained suggest that corn steep liquor contain substances that stimulate the synthesis of pigment and thus the cultivation with... (Complete abstract click electronic access below) / Mestre

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