Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa

fotografías a color, ilustraciones, gráficas, tablas

Autores:: Patiño Lagos, Margareth Andrea

Tipo de recurso:: Doctoral thesis

Fecha de publicación:: 2021

Institución:: Universidad Nacional de Colombia

Repositorio:: Universidad Nacional de Colombia

Idioma:

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repository_id_str
dc.title.spa.fl_str_mv	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
dc.title.translated.eng.fl_str_mv	Genetic improvement of a yeast Saccharomyces cerevisiae isolated in Colombia for xylose fermentation
title	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
spellingShingle	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa 570 - Biología 620 - Ingeniería y operaciones afines 660 - Ingeniería química 540 - Química y ciencias afines 500 - Ciencias naturales y matemáticas 600 - Tecnología (Ciencias aplicadas) Genetics Plant biotechnology Genética Biotecnología vegetal Saccharomyces cerevisiae Xilosa Producción de xilitol Recombinantes Silenciamiento génico ingeniería evolutiva Saccharomyces cerevisiae xylose xylitol production Recombinant Gene silencing Evolutionary engineering
title_short	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
title_full	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
title_fullStr	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
title_full_unstemmed	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
title_sort	Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa
dc.creator.fl_str_mv	Patiño Lagos, Margareth Andrea
dc.contributor.advisor.none.fl_str_mv	Velásquez Lozano, Mario Enrique Ugarte Stambuk, Boris Juan Carlos
dc.contributor.author.none.fl_str_mv	Patiño Lagos, Margareth Andrea
dc.contributor.researchgroup.spa.fl_str_mv	Grupo de Investigación en Procesos Químicos y Bioquímicos
dc.subject.ddc.spa.fl_str_mv	570 - Biología 620 - Ingeniería y operaciones afines 660 - Ingeniería química 540 - Química y ciencias afines 500 - Ciencias naturales y matemáticas 600 - Tecnología (Ciencias aplicadas)
topic	570 - Biología 620 - Ingeniería y operaciones afines 660 - Ingeniería química 540 - Química y ciencias afines 500 - Ciencias naturales y matemáticas 600 - Tecnología (Ciencias aplicadas) Genetics Plant biotechnology Genética Biotecnología vegetal Saccharomyces cerevisiae Xilosa Producción de xilitol Recombinantes Silenciamiento génico ingeniería evolutiva Saccharomyces cerevisiae xylose xylitol production Recombinant Gene silencing Evolutionary engineering
dc.subject.lemb.eng.fl_str_mv	Genetics Plant biotechnology
dc.subject.lemb.spa.fl_str_mv	Genética Biotecnología vegetal
dc.subject.proposal.spa.fl_str_mv	Saccharomyces cerevisiae Xilosa Producción de xilitol Recombinantes Silenciamiento génico ingeniería evolutiva
dc.subject.proposal.eng.fl_str_mv	Saccharomyces cerevisiae xylose xylitol production Recombinant Gene silencing Evolutionary engineering
description	fotografías a color, ilustraciones, gráficas, tablas
publishDate	2021
dc.date.issued.none.fl_str_mv	2021-04-19
dc.date.accessioned.none.fl_str_mv	2022-08-18T21:27:34Z
dc.date.available.none.fl_str_mv	2022-08-18T21:27:34Z
dc.type.spa.fl_str_mv	Trabajo de grado - Doctorado
dc.type.driver.spa.fl_str_mv	info:eu-repo/semantics/doctoralThesis
dc.type.version.spa.fl_str_mv	info:eu-repo/semantics/acceptedVersion
dc.type.coar.spa.fl_str_mv	http://purl.org/coar/resource_type/c_db06
dc.type.content.spa.fl_str_mv	Text
dc.type.redcol.spa.fl_str_mv	http://purl.org/redcol/resource_type/TD
format	http://purl.org/coar/resource_type/c_db06
status_str	acceptedVersion
dc.identifier.uri.none.fl_str_mv	https://repositorio.unal.edu.co/handle/unal/81969
dc.identifier.instname.spa.fl_str_mv	Universidad Nacional de Colombia
dc.identifier.reponame.spa.fl_str_mv	Repositorio Institucional Universidad Nacional de Colombia
dc.identifier.repourl.spa.fl_str_mv	https://repositorio.unal.edu.co/
url	https://repositorio.unal.edu.co/handle/unal/81969 https://repositorio.unal.edu.co/
identifier_str_mv	Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia
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dc.publisher.spa.fl_str_mv	Universidad Nacional de Colombia
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dc.publisher.department.spa.fl_str_mv	Instituto de Biotecnología (IBUN)
dc.publisher.faculty.spa.fl_str_mv	Facultad de Ciencias
dc.publisher.place.spa.fl_str_mv	Bogotá, Colombia
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spelling	Atribución-NoComercial-SinDerivadas 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Velásquez Lozano, Mario Enrique544472077e578088ab8dec69fe1e11bfUgarte Stambuk, Boris Juan Carlos93d8cc9e2976aa7904c509656c793a51Patiño Lagos, Margareth Andrea3063f3c1c18955548411911109657db6Grupo de Investigación en Procesos Químicos y Bioquímicos2022-08-18T21:27:34Z2022-08-18T21:27:34Z2021-04-19https://repositorio.unal.edu.co/handle/unal/81969Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/fotografías a color, ilustraciones, gráficas, tablasSaccharomyces cerevisiae es la principal levadura utilizada en biotecnología en todo el mundo, gracias a que su metabolismo y fisiología son conocidos permitiendo su aprovechamiento en diversos procesos industriales. Este microorganismo es excelente en la fermentación de azúcares como las hexosas, sin embargo, ha sido considerado como incapaz de metabolizar pentosas como la arabinosa y la xilosa presentes en la biomasa lignocelulósica. Esta biomasa es una materia prima ampliamente disponible que contiene xilosa, el segundo azúcar más abundante de la naturaleza, en aproximadamente el 35% de los azúcares totales. Esta fracción de azúcar podría ser aprovechada para la obtención de productos químicos de alto valor agregado como el xilitol. Utilizando ingeniería genética algunos investigadores han obtenido cepas recombinantes de S. cerevisiae con capacidad reducida de fermentar xilosa. El Grupo de Investigación en Procesos Químicos y Bioquímicos de la Universidad Nacional de Colombia realizó el aislamiento de algunos microorganismos obtenidos en Colombia, donde se identificó una cepa de S. cerevisiae, denominada como 202-3, que en presencia de hidrolizados lignocelulósicos mostró un consumo de xilosa del 2 % al 5%, característica que no se encuentra asociada a la especie. En esta investigación se confirmó mediante tres enfoques distintos que la cepa 202-3 efectivamente corresponde a una S. cerevisiae: mediante observación morfológica de la cepa por microscopía óptica y de barrido, por amplificación de un fragmento de 150 pb con iniciadores específicos para la especie, y por secuenciación de la región ITS. Su secuencia consenso mostró una similitud superior al 99 % respecto a las secuencias de S. cerevisiae reportadas en la base de datos genómica Blastn del NCBI. Experimentalmente, la cepa 202-3 no mostró un mejor consumo de xilosa que otras especies de levaduras analizadas consumidoras de esa pentosa, pero sí una metabolización significativa con un consumo del 9,8 %, lo cual hasta ahora no había sido reportado para ninguna cepa de S. cerevisiae. Sin embargo, para mejorar esa capacidad e intentar producir etanol a partir de la xilosa, la cepa 202-3 fue sometida a ingeniería genética y evolutiva. Determinada la ploidía de la cepa, se procedió a silenciar el gen GAL80 implicado en la represión de los genes GAL para que sean expresados continuamente y mejorar la captación y asimilación de esa pentosa. Para las recombinantes obtenidas los consumos de xilosa fueron de hasta el 18% con rendimiento de xilitol de hasta 0,407 g/g y no se obtuvo valores significativos de etanol. Mediante ingeniería evolutiva a la cepa parental y a dos recombinantes se obtuvo cepas mejoradas después de ocho inóculos sucesivos de 144 horas cada uno. De la cepa parental 202-3 que consumió 7% de xilosa, se obtuvo otra cepa que consumió 14% de xilosa y la producción de xilitol aumentó en 345% desde 0,236 g en el inóculo inicial a 1,050 g en el final. Con la cepa obtenida de la recombinante R2-MAPL (202-3, GAL80/gal80Δ::KanMX) el consumo de xilosa fue de 20% y la producción de xilitol aumentó 196% de 0,996 g inicial a 2,951 g final. Con la cepa obtenida de la recombinante B2G-MAPL (202-3, gal80Δ::KanMX/gal80Δ::Bler) el consumo final de xilosa fue de 28% y la producción de xilitol pasó de 1,115 g inicial a 4,876 g final representando un incremento de 337%. Estos resultados muestran que las estrategias utilizadas mejoraron el fenotipo de la cepa nativa 202-3 frente al consumo de xilosa y la producción de xilitol. (Texto tomado de la fuente)Saccharomyces cerevisiae is the most used yeast in biotechnology throughout the world because its metabolism, and physiology are well known, and it has been widely used in various industrial processes. This microorganism is excellent in the fermentation of sugars such as hexoses. However, it does not metabolize pentoses such as arabinose and xylose which are present in lignocellulosic biomass. This biomass constitutes a widely available raw material, with xylose content, the second most abundant sugar in nature, corresponding to approximately 35% of total sugars. This sugar fraction could be used to obtain high added value chemical products such as xylitol. Through genetic engineering some researchers have obtained recombinant yeast strains of S. cerevisiae with reduced xylose fermentation capability. The research group in Chemical and Biochemical Processes of the Universidad Nacional de Colombia performed the isolation of some microorganisms obtained in Colombia, where a S. cerevisiae strain called 202-3 was identified. This strain showed a xylose consumption between 2% and 5% in presence of lignocellulosic hydrolysates, characteristic that is not associated with the lineage. In this research, it was confirmed through three different approaches that the 202-3 strain is indeed a S. cerevisiae: morphologic strain observation by optical and SEM microscopy, amplification of a 150 bp fragment with species-specific primers, and sequencing its ITS region, whose consensus sequence showed similarity greater than 99% with the S. cerevisiae sequences reported in the NCBI Blastn genomic database. Experimentally, strain 202-3 did not show to be better than other yeast species with the capability to consume xylose, but it did show a significant metabolization of that pentose with a consumption of 9,8%, which until now had not been reported for S. cerevisiae. However, to improve that ability and try to produce ethanol from xylose, the strain was subjected to genetic and evolutionary engineering. Once the ploidy of the strain was determined, the GAL80 gene involved in the repression of the GAL genes was deleted so that they are continuously expressed and improve the uptake and assimilation of the pentose. For the recombinant strains obtained, the xylose consumptions were up to 18% with a xylitol yield of up to 0,407 g/g and no significant ethanol values were obtained. By evolutionary engineering to the parental strain and to two recombinants, improved stains were obtained after eight successive inoculum of 144 hours each. From the parental strain 202-3 that consumed 7% of xylose, another strain was obtained that consumed 14% of xylose and the xylitol production increased 345%, from 0,236 g in the initial inoculum to 1,050 g in the final one. With the strain obtained from recombinant R2-MAPL (202-3, GAL80/gal80Δ::KanMX), the xylose consumption was 20% and xylitol production increase 196%, from 0,996 g initial to 2,951 g final. With the strain obtained from recombinant B2G-MAPL (202-3, gal80Δ::KanMX/gal80Δ::Bler) the final xylose consumption was 28% and xylitol production went from 1,115 g initial to 4,876 g final, representing a 337% increase. These results show that the strategies used improved the phenotype of the native strain 202-3 against the consumption of xylose and the production of xylitol.DoctoradoDoctor en BiotecnologíaBioprocesos y Bioprospecciónxxviii, 163 páginasapplication/pdfUniversidad Nacional de ColombiaBogotá - Ciencias - Doctorado en BiotecnologíaInstituto de Biotecnología (IBUN)Facultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología620 - Ingeniería y operaciones afines660 - Ingeniería química540 - Química y ciencias afines500 - Ciencias naturales y matemáticas600 - Tecnología (Ciencias aplicadas)GeneticsPlant biotechnologyGenéticaBiotecnología vegetalSaccharomyces cerevisiaeXilosaProducción de xilitolRecombinantesSilenciamiento génicoingeniería evolutivaSaccharomyces cerevisiaexylosexylitol productionRecombinantGene silencingEvolutionary engineeringMejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosaGenetic improvement of a yeast Saccharomyces cerevisiae isolated in Colombia for xylose fermentationTrabajo de grado - Doctoradoinfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_db06Texthttp://purl.org/redcol/resource_type/TDColombiaAhmad, Irshad, Woo Yong Shim, y Jung-Hoe Kim. 2012. «Enhancement of xylitol production in glycerol kinase disrupted Candida tropicalis by co-expression of three genes involved in glycerol metabolic pathway». 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Applied and Environmental Microbiology 59 (5): 1487-94. https://doi.org/10.1128/AEM.59.5.1487-1494.1993EstudiantesInvestigadoresMaestrosLICENSElicense.txtlicense.txttext/plain; charset=utf-84074https://repositorio.unal.edu.co/bitstream/unal/81969/2/license.txt8153f7789df02f0a4c9e079953658ab2MD52ORIGINAL1093752481.2021.2.pdf1093752481.2021.2.pdfTesis de Doctorado en Biotecnologíaapplication/pdf6519561https://repositorio.unal.edu.co/bitstream/unal/81969/3/1093752481.2021.2.pdf5b6f1124b86e26bbfd663330bac951a4MD53THUMBNAIL1093752481.2021.2.pdf.jpg1093752481.2021.2.pdf.jpgGenerated Thumbnailimage/jpeg5359https://repositorio.unal.edu.co/bitstream/unal/81969/4/1093752481.2021.2.pdf.jpg7f12907cc422a700430f928445eec442MD54unal/81969oai:repositorio.unal.edu.co:unal/819692024-08-08 23:12:01.508Repositorio Institucional Universidad Nacional de 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EVESURBIFBPUiBMQSBTRUNSRVRBUsONQSBHRU5FUkFMLiAqTEEgVEVTSVMgQSBQVUJMSUNBUiBERUJFIFNFUiBMQSBWRVJTScOTTiBGSU5BTCBBUFJPQkFEQS4gCgpBbCBoYWNlciBjbGljIGVuIGVsIHNpZ3VpZW50ZSBib3TDs24sIHVzdGVkIGluZGljYSBxdWUgZXN0w6EgZGUgYWN1ZXJkbyBjb24gZXN0b3MgdMOpcm1pbm9zLiBTaSB0aWVuZSBhbGd1bmEgZHVkYSBzb2JyZSBsYSBsaWNlbmNpYSwgcG9yIGZhdm9yLCBjb250YWN0ZSBjb24gZWwgYWRtaW5pc3RyYWRvciBkZWwgc2lzdGVtYS4KClVOSVZFUlNJREFEIE5BQ0lPTkFMIERFIENPTE9NQklBIC0gw5psdGltYSBtb2RpZmljYWNpw7NuIDE5LzEwLzIwMjEK

Mejoramiento genético de una levadura Saccharomyces cerevisiae aislada en territorio colombiano para la fermentación de xilosa

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