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Optimización De La Producción De Riboflavina En Ashbya Gossypii Basada En El Análisis Funcional De Las Rutas De Recuperación De Nucleobases De Purina

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Optimización De La Producción De Riboflavina En Ashbya Gossypii Basada En El Análisis Funcional De Las Rutas De Recuperación De Nucleobases De Purina Universidad de Salamanca Departamento de Microbiología y Genética Optimización de la producción de riboflavina en Ashbya gossypii basada en el análisis funcional de las rutas de recuperación de nucleobases de purina. José Antonio Uña Álvarez Tesis Doctoral 2013 Dr. Luís Román Fernández Lago, Director del Departamento de Microbiología y Genética de la Universidad de Salamanca. CERTIFICA: Que la memoria titulada “Optimización de la producción de riboflavina en Ashbya gossypii basada en el análisis funcional de las rutas de recuperación de nucleobases de purina”, presentada por D. José Antonio Uña Álvarez para optar al grado de Doctor en Biología, ha sido realizada bajo la dirección de la Dra. Mª Ángeles Santos García y del Dr. José Luis Revuelta Doval, en el Departamento de Microbiología y Genética de la Universidad de Salamanca. Y para que así conste expide el presenta certificado en: Salamanca a de de 2013 Fdo. Dr. Luís Román Fernández Lago Dra. Mª Ángeles Santos García, Profesora Titular del Departamento de Microbiología y Genética de la Universidad de Salamanca y Dr. José Luis Revuelta Doval, Catedrático de Genética del Departamento de Microbiología y Genética de la Universidad de Salamanca. CERTIFICAN: Que el doctorando José Antonio Uña Álvarez ha realizado en el Departamento de Microbiología y Genética de la Universidad de Salamanca, bajo su dirección, el trabajo titulado “Optimización de la producción de riboflavina en Ashbya gossypii basada en el análisis funcional de las rutas de recuperación de nucleobases de purina”, para optar al grado de Doctor en Biología. Y para que así conste, y como directores del trabajo, expiden el presente certificado en: Salamanca a de de 2013 Fdo: Dra. Mª Ángeles Santos García. Fdo. Dr. José Luis Revuelta Doval A mis padres. Ninguna tesis se escribe en total soledad, ni es fruto de un solo par de manos, ni de una sola mente. Tampoco se deben olvidar todos aquellos hombros que han cargado con tanto peso durante este tiempo. Unas breves líneas no harán justicia de todo lo que se debe agradecer. Pero aspiro en el día a días a que esa justicia sea hecha. Agradezco: A los directores de este trabajo: Dra. Santos y Dr. Revuelta por darme la oportunidad de desarrollar este trabajo y la dirección y determinación con la que lo habéis seguido. Y también por darme a conocer de primera mano cómo es ser un biólogo de bata. A la Junta de Castilla y León y Fondo Social Europeo por la concesión de la Beca para la realización de esta Tesis Doctoral. Y por la financiación de este trabajo mediante el Proyecto Ref. SA098A09 de la Consejería de Educación de la Junta de Castilla y León. A mis compañeros de laboratorio tanto pasados como presentes: Fernando, Alberto, Rubén, Rodrigo, José Miguel, Rafa y las niñas: Patri, Cris, Lola, Silvia, Sole, Patri la Nueva y Cris (no, no cambio y no os voy a distinguir por el nombre). Porque de todos es un pedacito de esta tesis. Especialmente a Angie por creer en mis capacidades; y aunque a veces tengamos puntos de vista diferentes el objetivo no cambia. A mis compañeros de facultad, especialmente a los que siguen perdidos por aquí o casi: Guille, Javi, Charli, Ana, Abe, Pi, Raúl (todavía una caja de puntas tiene tu nombre…) que tantas horas y vicios hemos compartido. A la gente del departamento, tanto los que siguen en estas tierras como los que colonizaron otras nuevas, por las tertulias de las horas de comer, cenas y esas pachanguitas de baloncesto tan buenas (prometo que volveré a jugar). Especialmente a Pedrito que además es mi rastreador de artículos raros. A mis compañeros del Insycal, que aunque este año nos quedáramos sin jugar, seguimos siendo más equipo fuera de la cancha que dentro. A los de siempre Señor Rober, Ale, Jorgito, Chema, con vosotros los problemas se diluyen. Bicha lo tuyo es trampa que te has ido pa tierras mu lejanas. A mi familia, mis padres, mi herma, tíos, primos, abuelos que habéis estado pendientes en todo momento ¿Y cuándo no? Porque con vuestro apoyo habéis hecho que esto sea posible. ABREVIATURAS 2,8 -DHA 2,8 -dihidroxiadenina ADP Adenosina-5'-difosfato Ag Ashbya gossypii AICAR 5-aminoimidazol 4-carboxamida ribonucleótido AICAR 5-aminoimidazol 4-carboxamida AIR 5-aminoimidazol ribonucleótido AK adenina quinasa AMP Adenosina-5'-monofosfato APRT adenina fosforribosiltransferasa ATP Adenosina-5'-trifosfato dominio de regulación e interacción de Bas1p (de la terminología BIRD inglesa “Bas1p Interaction Regulador Domain”) secuencia de reconocimiento de Bas1p (de la terminología inglesa BRE “Bas1 Recognition Element”) CAIR carboxiaminoimidazol ribótido CDR cantidad diaria recomendada dominio de unión a ADN (de la terminología inglesa “DNA Binding DBD Domain”) DMT Drug Metabolite Transporter ENT Equilibrative Nucleoside Transporter FAD dinucleótido de flavina y adenina FAICAR 5-formaminoimidazol 4-carboxamida ribonucleótido FGAM formilglicinamidina ribonucleótido FGAR formil glicinamida ribonucleótido FMN mononucleótido de flavina GAR glicinamida ribonucleótido GDP Guanosina-5'-difosfato GK guanina quinasa GMP Guanosina-5'-monofosfato GPRT guanina fosforribosiltransferasa GRE elemento regulador en presencia de guanina GTP Guanosina-5'-trifosfato HGPRT hipoxantina guanina fosforribosiltransferasa HGXPRT hipoxantina guanina xantina fosforribosiltransferasa HPRT hipoxantina fosforribosiltransferasa IMP Inosina-5'-monofosfato ISN nucleotidasa específica de IMP MPA Ácido micofenólico NAT Nucleobase Ascorbate Transporter NCS2 Nucleobase Cation Symporter 2 NSE elemento regulador en situación limitante de nutrientes PNP nucleosido fosforilasa de purinas PRA 5- fosforribosilamina PRPP 5-fosforribosil-1-pirofosfato PRT fosforribosiltransferasa PUP Purine Uptake Permease RFK quinasa de riboflavina reacción en cadena de la polimerasa a tiempo real (de la RT-PCR terminología inglesa “Real Time-Polymerase Chaín Reaction”). SAICAR 6-aminoimidazol 4-N-succinicocarboxamida ribonucleótido Sc Saccharomyces cerevisiae THF tetrahidrofolato UPS Ureidin Permease XMP xantosina-5'-monofosfato XPRT xantina fosforribosiltransferasa Índice INTRODUCCIÓN I. LA RIBOFLAVINA O VITAMINA B2. ........................................................................................... 1 I.1. ESTRUCTURA Y FUNCIÓN BIOLÓGICA. ................................................................................................. 1 I.2. NUTRICIÓN Y FARMACOLOGÍA. ................................................................................................ 2 II. EL HONGO SOBREPRODUCTOR DE RIBOFLAVINA ASHBYA GOSSYPII. .......................... 5 II.1 TAXONOMÍA Y BIOLOGÍA ........................................................................................................... 5 II.2. BIOSÍNTESIS Y PRODUCCIÓN DE RIBOFLAVINA .................................................................. 7 II.2.1. Ruta biosintética de la riboflavina ............................................................................................ 9 II.2.2. Biosíntesis del precursor GTP ................................................................................................ 11 II.2.2.1. Ruta de síntesis de novo de nucleótidos de purina ........................................................... 11 II.2.2.2. Biosíntesis de nucleótidos de purina a través de las rutas de recuperación .................... 14 II.2.2.3. Regulación de la biosíntesis de nucleótidos de purina ..................................................... 19 II.2.2.4. Farmacología y biosíntesis de purinas ............................................................................. 22 II.2.3. Ashbya gossypii en la producción industrial de riboflavina ................................................... 23 OBJETIVOS OBJETIVOS ....................................................................................................................................... 29 RESULTADOS I. DISECCIÓN GENÉTICA DE LAS RUTAS DE RECUPERACION DE NUCLEOBASES DE PURINA EN A. GOSSYPII ................................................................................................................. 31 I.1. GENES IMPLICADOS EN LAS RUTAS DE RECUPERACIÓN .................................................. 31 I.1.1. Comparación de genomas: análisis de homología. .................................................................. 31 I.1.2. Estudios de complementación heteróloga ................................................................................ 34 I.1.2.1. Rutas de recuperación de adenina ..................................................................................... 34 I.1.2.2. Rutas de recuperación de xantina y guanina ..................................................................... 37 I.2. ANÁLISIS FUNCIONAL ............................................................................................................... 40 I.2.1. Inactivación de los genes AgAPT1,AgXPT1 y AgAMD1 .......................................................... 40 I.2.2. Actividades enzimáticas............................................................................................................ 44 I.2.3. Modulación de las actividades enzimáticas ............................................................................. 45 I.2.4. Relación funcional de las rutas de recuperación y la ruta de novo ......................................... 47 I.2.5. Recuperación de la nucleobase no convencional AICA ........................................................... 49 I.2.6. Localización subcelular de las enzimas implicadas en las rutas de recuperación ..................
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