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Toxina PR En Penicillium Roqueforti

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Toxina PR En Penicillium Roqueforti Universidad de León Instituto de Biotecnología de León Dpto. de Biología Molecular INBIOTEC Área de Microbiología TESIS DOCTORAL Caracterización Bioquímica y Molecular de la Biosíntesis del Antitumoral Andrastina y de la Toxina PR en Penicillium roqueforti . Pedro Iván Hidalgo Yanes León 2013 INFORME DEL DIRECTOR DE LA TESIS 1 (R.D. 99/2011, de 28 de enero y Normativa de la ULE) El Dr. D. Juan Francisco Martín Martín, el Dr. D. Ricardo Vicente Ullán y la Dra. Dña. Silvia Albillos García como Directores 2 de la Tesis Doctoral titulada “Caracterización Bioquímica y Molecular de la Biosíntesis del Antitumoral Andrastina y de la Toxina PR en Penicillium roqueforti ” realizada por D. Pedro Iván Hidalgo Yanes en el programa de doctorado Biología Molecular y Biotecnología, informan favorablemente el depósito de la misma, dado que reúne las condiciones necesarias para su defensa. León a ___ de__________ de _____________ D. Juan Francisco Martín Martín D. Ricardo Vicente Ullán Dña. Silvia Albillos García 1 Este impreso solamente se cumplimentará para los casos de tesis depositadas en papel. 2 Si la Tesis está dirigida por más de un Director tienen que constar los datos de cada uno y han de firmar todos ellos. ADMISIÓN A TRÁMITE DE LA TESIS DOCTORAL 3 (R.D. 99/2011, de 28 de enero y Normativa de la ULE) El órgano responsable del programa de doctorado _____________________________ ___________________________________________________________________________ en su reunión celebrada el día ___ de _____________ de ________ ha acordado dar su conformidad a la admisión a trámite de lectura de la Tesis Doctoral titulada “Caracterización Bioquímica y Molecular de la Biosíntesis del Antitumoral Andrastina y de la Toxina PR en Penicillium roqueforti ”, dirigida por el Dr. D. Juan Francisco Martín Martín, el Dr. D. Ricardo Vicente Ullán y la Dra. Dña. Silvia Albillos García, elaborada por D. Pedro Iván Hidalgo Yanes.y cuyo título en inglés es el siguiente “Biochemical and Molecular Characterization of the Antitumoral Andratine and PR-toxin in Penicillium roqueforti ”. Lo que firmo, en León a _____ de ______________ de ___________. El Secretario del Departamento/ Secretario de la Comisión Académica, Fdo.: _________________ CONFORMIDAD El Director del Departamento/ Presidente de la Comisión Académica, Fdo.: ____________________ 3 Este impreso solamente se cumplimentará para los casos de tesis depositadas en papel. A mis padres Teresa y Pedro Pablo y a mi hermana Idania Agradecimientos Quiero expresar mi agradecimiento a las siguientes instituciones y personas sin las cuales hubiera sido imposible el desarrollo de esta memoria de Tesis Doctoral. Al Dr. Juan Francisco Martín, director de esta Tesis, por ofrecerme la oportunidad de trabajar bajo su tutela y por el apoyo brindado para lograr la culminación de este trabajo. A la Agencia Española de Colaboración Internacional para el Desarrollo (AECID) por la financiación de los dos primeros años de mi estancia de investigación. Al Dr. Ricardo Vicente Ullán y la Dra. Silvia M. Albillos García, co-directores de esta Tesis, por haberme guiado en las distintas fases de la realización de este trabajo y por el aliento para continuar en los momentos más difíciles. Al Dr. Carlos Barreiro Méndez por su colaboración y asesoría brindada en los trabajos de proteómica. Al Dr. Olimpio Montero Domínguez por colaborar en la realización de las determinaciones por espectrometría de masas. Al Dr. Carlos García Estrada por brindarme su apoyo y asesoramiento. A Marta y Fernando, compañeros de cuarto, por compartir los momentos de alegría y de frustración propios del proceso de investigación. A Mafe, Rebeca, Raquel, Vanesa, Miriam, Carlos y Lorena por haberme acogido y hacerme sentir en INBIOTEC como en casa. A todos los compañeros con los que he coincidido durante toda o parte de mi estancia en INBIOTEC por haber creado un ambiente de trabajo propicio para el desarrollo de la investigación científica. Y finalmente deseo expresar mi gratitud a todas aquellas personas que de un modo u otro han contribuido a la realización de esta tesis doctoral. La ignorancia afirma o niega rotundamente; la ciencia duda. Voltaire Índice General Introducción .. 3 1. Características generales del género Penicillium .... 3 2. Características generales de Penicillium roqueforti .... 5 2.1 Características taxonómicas.......... 6 3. Papel de P. roqueforti en la elaboración del queso azul. Produc ción de enzimas extracelulares..... 9 4. Micotoxinas producidas por P. roqueforti .. 11 5. Metabolismo secundario de P. roqueforti ..... 13 5.1 Metabolismo de la toxina PR...... 14 5.1.1 Aislamiento y caracterización de la toxi na PR y compuestos afines............................................................................................................... 14 5.1.2 Biosíntesis de la toxina PR..... 16 5.1.3 Aristoloqueno, posible molécula precursora para la biosíntesis de la toxina PR...... 18 5.1.4 Características generales de la aristoloqueno sintasa Ari1, enzima responsable de la biosíntesis de aristoloqueno 20 5.1.5 Metabolitos secundarios resultantes del proceso de transforma ción de la toxina PR 22 5.1.6 Perfil quimiotaxonómico de las cepas productoras de toxina PR.. 24 5.2 Metabolismo de las andrastinas 26 5.2.1 Aislamiento y estructura química... 26 5.2.2 Actividad biológica y mecanismo de acción 27 5.2.3 Ruta biosintética de la andrastina A.. 29 5.3 Metabolismo del ácido micofenólico 29 5.3.1 Asilamiento e identificación del ácido micofenólico... 29 5.3.2 Propiedades biológicas y mecanismo de acción del ácido micofenólico... 30 5.3.3 Biosíntesis del ácido micofenólico 31 6. Diseño de Experimentos en estudios de fermentación.... 33 6.1 Diseño factorial. 33 6.2 Diseño de superficies de respuesta..... 34 Objetivos .... 39 Materiales y Métodos .. 43 1. Microorganismos 43 1.1 Cepas bacterianas. 43 1.2 Cepas fúngicas... 43 2. Vectores.. 43 3. Reactivos químicos 44 3.1 Antibióticos.. 44 3.2 Enzimas. 44 3.3 Oligonucleótidos.. 45 3.4 Conjuntos de Kits comerciales.. 46 4. Medios de Cultivo.. 46 4.1 Medios de cultivo para bacterias... 46 4.2 Medios de cultivo para hongos filamentosos.. 47 5. Crecimiento y mantenimiento de microorganismos.. 54 5.1 Cultivos bacterianos... 54 5.2 Cultivos fúngicos. 55 6. Producción de metabolitos secundarios en P. roqueforti . 56 6.1 Extracción de andrastinas a partir de cultivos en medio sólido 56 6.2 Extracción de andrastinas a partir de cultivos en medio líquido.. 56 6.3 Extracción de toxina PR a partir de cultivos en medio líquido.. 57 6.4 Extracción de toxina PR a partir del cultivo de P. roqueforti en arroz. 57 6.5 Cuantificación volumétrica de andrastinas mediante HPLC. 58 6.6 Cuantificación volumétrica de ácido micofenólico. 58 6.7 Identificación cromatográfica de toxina PR. 59 6.8 Cuantificación relativa de toxina PR. 59 7. Obtención de ADN total de P. roqueforti a partir de micelio. 60 8. Manipulación de ADN 61 8.1 Eliminación selectiva de ácidos nucleicos... 61 8.2 Limpieza y precipitación de ADN. 61 8.3 Reacciones con endonucleasas de restricción... 62 8.4 Ligación de fragmentos de ADN.. 63 8.5 Reacción en cadena de la polimerasa (PCR). 64 8.6 Electroforesis de ADN en geles de agarosa 66 8.7 Extracción de ADN de geles de agarosa. 68 8.8 Cuantificación y análisis de pureza de los ácidos nucleicos. 68 9. Transformación de E. coli y obtención de ADN plasmídico. 69 9.1 Obtención de células de E. coli competentes. 69 9.2 Transformación de E. coli . 70 9.3 Minipreparación de ADN plasmídico 70 9.4 Aislamiento de ADN plasmídico de E. coli a gran escala.. 71 10. Transferencia de ADN de geles de agarosa a filtros de nailo n (Southern blotting) 71 10.1 Transferencia de ADN mediante sistema de vacío.. 72 11. Hibridación no radioactiva de ADN 73 11.1 Marcaje de sondas de ADN. 73 11.2 Prehibridación 73 11.3 Hibridación. 74 11.4 Lavado de los filtros. 74 11.5 Detección Inmunológica.. 75 12. Obtención de mutantes por luz ultravioleta.. 76 13. Transformación de P. roqueforti . 76 13.1 Obtención de protoplastos de P. roqueforti... 77 13.2 Transformación de protoplastos. 78 14. Caracterización fenotípica.. 79 14.1 Extensión radial (apical) 79 14.2 Peso seco en medio líquido. 79 15. Métodos para análisis de ARN.. 80 15.1 Obtención de ARN total a gran escala.. 80 15.2 Tratamientos con ADNasas de las muestras de ARN. 81 15.3 Transcripción reversa de ARN (RT-PCR) 82 16. Métodos para el análisis de proteínas... 84 16.1 Extracción de proteínas de P. roqueforti 84 16.2 Determinación de la concentración de proteínas. 85 16.3 Electroforesis de proteínas en condiciones desnaturalizantes.. 86 16.4 Análisis bidimensional de proteínas de P. roqueforti ... 87 16.4.1 Primera dimensión o isoelectroenfoque (IEF).. 87 16.4.2 Segunda dimensión (SDS-PAGE).... 88 16.5 Detección de proteínas 90 16.5.1 Tinción por el método de Coomassie Coloidal..... 91 16.5.2 Tinción por DIGE. 91 16.6 Digestión tríptica de proteínas separadas mediante electroforesis 2D- PAGE. 92 17. Aplicación de diseño de experimento 93 Capítulo I. Estudio de la producción de andrastina A en Penicillium roqueforti 95 Objetivos del Capítulo I .. 99 Resultados del Capítulo I .. 103 1. Optimización de la producción de andrastinas en P. roqueforti CECT 2905 103 1.1 Experimentos previos. 103 1.2 Primer diseño “factorial 3 3” 106 1.3 Segundo diseño “factorial 3 3” 111 2. Obtención de mutantes de P. roqueforti CECT 2905 con la producción de andrastina A afectada 117 3. Evaluación fenotípica de los mutantes 119 3.1 Evaluación fenotípica de los mutantes en medio sólido... 119 3.2 Evaluación fenotípica de los mutantes en medio líquido.. 121 4. Estudio proteómico comparativo entre los mutantes afectados en la producción de andrastina A y la cepa parental P. roqueforti CECT 2905.. 123 Discusión del Capítulo I .
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