Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações do UNIOESTE |
Texto Completo: | http://tede.unioeste.br/handle/tede/3481 |
Resumo: | Second generation bioethanol employs lignocellulosic materials in its preparation. One of the steps for these materials degradation utilizes cellulases produced by microorganisms. Among these, Trichoderma reesei fungus is one of the main cellulases producers used in industry. This fungus genetic modification can lead to enzimes production optimization, reducing cost and improving biofuels manufacture. Thus, the present work objective was delete the sequence encoding zinc fingers motifs of cellulase ACE1repressor transcription factor from T. reesei RUT-C30 fungus, seeking enzymatic production optimization. In primers construction for amplification ACE1 regions 5’ and 3' and the hph selection marker, which confers hygromycin B resistance, Joint Genome Institute - JGI site and the BioEdit ® program were used. The deletion cassette with pRS426 vector construction was mediated by Saccharomyces cerevisiae SC9721 yeast. After the cassette construction, T. reesei RUT-C30 transformation was made by protoplast and this transformation confirmation was effected by part of the hph using hphNestF and hphNestR amplification primers. After transformation with mutants obtained, endoglucanase, exoglucanase and total cellulase activity was quantified with carboxymethylcellulose substrates (CMC), microcrystalline cellulose (Avicel®) and Whatman paper filter (PF), respectively. The enzymatic production and biomass hydrolysis efficiency were performed comparing RUT-C30 strain for mutants. After deletion cassette construction, a 3501 bp fragment amplification confirmed the cassette formation. Posteriorly, RUT-C30 strain transformation, a 989 bp amplification was observed, confirming the 3 mutants target sequence deletion. With cellulase activity assay, 3 transformed strain showed higher enzymatic production when compared to RUT-C30 strain. In this comparison, a significant statistical difference was observed of RUT-C30Δace1-1 strain with Avicel® and PF (p <0.001) CMC (p <0.01), RUT-C30Δace1-2 strain with CMC (p<0,01) e PF (p<0,05), and RUT-C30Δace1-3 strain with Avicel (p<0,001), CMC and PF (p<0,01). The mutants also showed greater efficiency in biomass hydrolysis, with release sugar increase between 21 and 42%. Based on this study, mutants are promising for most efficient and viable ethanol production. Nevertheless, additional tests must be carried out to better understand these fungi applicability in the industrial level. |
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Maller, Alexandrehttp://lattes.cnpq.br/8153318875076127da Conceição Silva, José Luishttp://lattes.cnpq.br/9251091711372741Maller, Ana Cláudia Paiva Alegrehttp://lattes.cnpq.br/8189048974419765http://lattes.cnpq.br/9062171151802553Dudek, Débora Nakadomari2018-03-05T19:50:05Z2017-02-07DUDEK, Débora Nakadomari. Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30. 2017. 56 f. Dissertação (Mestrado - Programa de Pós-Graduação em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel, 2017.http://tede.unioeste.br/handle/tede/3481Second generation bioethanol employs lignocellulosic materials in its preparation. One of the steps for these materials degradation utilizes cellulases produced by microorganisms. Among these, Trichoderma reesei fungus is one of the main cellulases producers used in industry. This fungus genetic modification can lead to enzimes production optimization, reducing cost and improving biofuels manufacture. Thus, the present work objective was delete the sequence encoding zinc fingers motifs of cellulase ACE1repressor transcription factor from T. reesei RUT-C30 fungus, seeking enzymatic production optimization. In primers construction for amplification ACE1 regions 5’ and 3' and the hph selection marker, which confers hygromycin B resistance, Joint Genome Institute - JGI site and the BioEdit ® program were used. The deletion cassette with pRS426 vector construction was mediated by Saccharomyces cerevisiae SC9721 yeast. After the cassette construction, T. reesei RUT-C30 transformation was made by protoplast and this transformation confirmation was effected by part of the hph using hphNestF and hphNestR amplification primers. After transformation with mutants obtained, endoglucanase, exoglucanase and total cellulase activity was quantified with carboxymethylcellulose substrates (CMC), microcrystalline cellulose (Avicel®) and Whatman paper filter (PF), respectively. The enzymatic production and biomass hydrolysis efficiency were performed comparing RUT-C30 strain for mutants. After deletion cassette construction, a 3501 bp fragment amplification confirmed the cassette formation. Posteriorly, RUT-C30 strain transformation, a 989 bp amplification was observed, confirming the 3 mutants target sequence deletion. With cellulase activity assay, 3 transformed strain showed higher enzymatic production when compared to RUT-C30 strain. In this comparison, a significant statistical difference was observed of RUT-C30Δace1-1 strain with Avicel® and PF (p <0.001) CMC (p <0.01), RUT-C30Δace1-2 strain with CMC (p<0,01) e PF (p<0,05), and RUT-C30Δace1-3 strain with Avicel (p<0,001), CMC and PF (p<0,01). The mutants also showed greater efficiency in biomass hydrolysis, with release sugar increase between 21 and 42%. Based on this study, mutants are promising for most efficient and viable ethanol production. Nevertheless, additional tests must be carried out to better understand these fungi applicability in the industrial level.O bioetanol de segunda geração emprega materiais lignocelulósicos na sua elaboração. Uma das etapas para a degradação destes materiais utiliza celulases produzidas por microrganismos. Dentre estes, o fungo Trichoderma reesei é um dos principais produtores de celulases utilizadas na indústria. A modificação genética deste fungo pode levar à otimização da produção de suas enzimas, diminuindo o custo e melhorando a fabricação de biocombustíveis. Desta forma, o objetivo do trabalho foi deletar a região dos motivos dedos de zinco no gene que codifica o fator de transcrição repressor de celulase ACE1 do fungo T. reesei RUT-C30, buscando a otimização na produção enzimática. Na construção dos primers para amplificação das regiões 5’ e 3’ de ace1 e do marcador de seleção hph, que confere resistência à higromicina B, utilizou-se o site Joint Genome Institute – JGI e o programa BioEdit®. A construção do cassete de deleção com o vetor pRS426 foi mediado pela levedura Saccharomyces cerevisiae SC9721. Posteriormente, a construção do cassete, a transformação de T. reesei RUT-C30 foi realizada através de protoplasto e a confirmação desta transformação foi efetuada por amplificação de parte do hph utilizando os primers hphNestF e hphNestR. Após a transformação, com os mutantes obtidos, a atividade de endoglucanase, exoglucanase e celulase total foi quantificada com os substratos carboximetilcelulose (CMC), celulose microcristalina (Avicel®) e papel de filtro Whatman (PF), respectivamente. A produção enzimática e a eficiência na hidrólise da biomassa foram realizadas comparando-se a linhagem RUT-C30 aos mutantes. Após a construção do cassete de deleção, a amplificação de um fragmento de 3501 pb confirmou a formação do cassete. E, posteriormente à transformação da linhagem RUT-C30, o amplificado de 989 pb foi observado, confirmando a deleção da sequência alvo em 3 mutantes. Com o ensaio de atividade de celulases, as 3 linhagens transformadas mostraram maior produção enzimática quando comparadas à linhagem RUT-C30. Nessa comparação, foi observada diferença estatística significativa da linhagem RUT-C30Δace1-1 com Avicel® e PF (p<0,001), da linhagem RUTC30Δace1- 2 com CMC (p<0,01) e PF (p<0,05) e da linhagem RUT-C30Δace1-3 com Avicel (p<0,001), CMC e PF (p<0,01). Os mutantes também apresentaram maior eficiência na hidrólise da biomassa, com aumento na liberação de açúcar entre 21 e 42%. Com base nos dados deste estudo, os mutantes apresentam-se promissores para a produção mais eficiente e viável de etanol. Apesar disso, testes adicionais devem ser realizados para melhor entendimento da aplicabilidade destes fungos a nível industrial.Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2018-03-05T19:50:05Z No. of bitstreams: 2 Debora_Dudek2017.pdf: 1099947 bytes, checksum: eb7356b2bdfdfe7d3d4da8e48d12d05c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-03-05T19:50:05Z (GMT). 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dc.title.por.fl_str_mv |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
dc.title.alternative.eng.fl_str_mv |
Partial deletion of ACE1 transcription factor for optimization Trichoderma reesei RUT-C30 cellulase production |
title |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
spellingShingle |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 Dudek, Débora Nakadomari Fungo Cassete de deleção Transformação Material lignocelulósico Bioetanol Fungus Deletion cassette Transformation Lignocellulosic material Bioethanol CIENCIAS DA SAUDE::FARMACIA |
title_short |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
title_full |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
title_fullStr |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
title_full_unstemmed |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
title_sort |
Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30 |
author |
Dudek, Débora Nakadomari |
author_facet |
Dudek, Débora Nakadomari |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Maller, Alexandre |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/8153318875076127 |
dc.contributor.referee1.fl_str_mv |
da Conceição Silva, José Luis |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/9251091711372741 |
dc.contributor.referee2.fl_str_mv |
Maller, Ana Cláudia Paiva Alegre |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/8189048974419765 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/9062171151802553 |
dc.contributor.author.fl_str_mv |
Dudek, Débora Nakadomari |
contributor_str_mv |
Maller, Alexandre da Conceição Silva, José Luis Maller, Ana Cláudia Paiva Alegre |
dc.subject.por.fl_str_mv |
Fungo Cassete de deleção Transformação Material lignocelulósico Bioetanol |
topic |
Fungo Cassete de deleção Transformação Material lignocelulósico Bioetanol Fungus Deletion cassette Transformation Lignocellulosic material Bioethanol CIENCIAS DA SAUDE::FARMACIA |
dc.subject.eng.fl_str_mv |
Fungus Deletion cassette Transformation Lignocellulosic material Bioethanol |
dc.subject.cnpq.fl_str_mv |
CIENCIAS DA SAUDE::FARMACIA |
description |
Second generation bioethanol employs lignocellulosic materials in its preparation. One of the steps for these materials degradation utilizes cellulases produced by microorganisms. Among these, Trichoderma reesei fungus is one of the main cellulases producers used in industry. This fungus genetic modification can lead to enzimes production optimization, reducing cost and improving biofuels manufacture. Thus, the present work objective was delete the sequence encoding zinc fingers motifs of cellulase ACE1repressor transcription factor from T. reesei RUT-C30 fungus, seeking enzymatic production optimization. In primers construction for amplification ACE1 regions 5’ and 3' and the hph selection marker, which confers hygromycin B resistance, Joint Genome Institute - JGI site and the BioEdit ® program were used. The deletion cassette with pRS426 vector construction was mediated by Saccharomyces cerevisiae SC9721 yeast. After the cassette construction, T. reesei RUT-C30 transformation was made by protoplast and this transformation confirmation was effected by part of the hph using hphNestF and hphNestR amplification primers. After transformation with mutants obtained, endoglucanase, exoglucanase and total cellulase activity was quantified with carboxymethylcellulose substrates (CMC), microcrystalline cellulose (Avicel®) and Whatman paper filter (PF), respectively. The enzymatic production and biomass hydrolysis efficiency were performed comparing RUT-C30 strain for mutants. After deletion cassette construction, a 3501 bp fragment amplification confirmed the cassette formation. Posteriorly, RUT-C30 strain transformation, a 989 bp amplification was observed, confirming the 3 mutants target sequence deletion. With cellulase activity assay, 3 transformed strain showed higher enzymatic production when compared to RUT-C30 strain. In this comparison, a significant statistical difference was observed of RUT-C30Δace1-1 strain with Avicel® and PF (p <0.001) CMC (p <0.01), RUT-C30Δace1-2 strain with CMC (p<0,01) e PF (p<0,05), and RUT-C30Δace1-3 strain with Avicel (p<0,001), CMC and PF (p<0,01). The mutants also showed greater efficiency in biomass hydrolysis, with release sugar increase between 21 and 42%. Based on this study, mutants are promising for most efficient and viable ethanol production. Nevertheless, additional tests must be carried out to better understand these fungi applicability in the industrial level. |
publishDate |
2017 |
dc.date.issued.fl_str_mv |
2017-02-07 |
dc.date.accessioned.fl_str_mv |
2018-03-05T19:50:05Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
DUDEK, Débora Nakadomari. Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30. 2017. 56 f. Dissertação (Mestrado - Programa de Pós-Graduação em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel, 2017. |
dc.identifier.uri.fl_str_mv |
http://tede.unioeste.br/handle/tede/3481 |
identifier_str_mv |
DUDEK, Débora Nakadomari. Deleção parcial do fator de transcrição ACE1 para otimização da produção de celulases por Trichoderma reesei RUT-C30. 2017. 56 f. Dissertação (Mestrado - Programa de Pós-Graduação em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel, 2017. |
url |
http://tede.unioeste.br/handle/tede/3481 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.por.fl_str_mv |
UNIOESTE, CAPES e FUNDAÇÃO ARAUCÁRIA |
dc.relation.program.fl_str_mv |
7878055067573953101 |
dc.relation.confidence.fl_str_mv |
600 600 600 600 |
dc.relation.department.fl_str_mv |
-8940439713387849267 |
dc.relation.cnpq.fl_str_mv |
6997636413449754996 |
dc.relation.sponsorship.fl_str_mv |
-2555911436985713659 |
dc.rights.driver.fl_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Ciências Farmacêuticas |
dc.publisher.initials.fl_str_mv |
UNIOESTE |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Centro de Ciências Médicas e Farmacêuticas |
publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações do UNIOESTE instname:Universidade Estadual do Oeste do Paraná (UNIOESTE) instacron:UNIOESTE |
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Universidade Estadual do Oeste do Paraná (UNIOESTE) |
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UNIOESTE |
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UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE |
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http://tede.unioeste.br:8080/tede/bitstream/tede/3481/5/Debora_Dudek2017.pdf http://tede.unioeste.br:8080/tede/bitstream/tede/3481/2/license_url http://tede.unioeste.br:8080/tede/bitstream/tede/3481/3/license_text http://tede.unioeste.br:8080/tede/bitstream/tede/3481/4/license_rdf http://tede.unioeste.br:8080/tede/bitstream/tede/3481/1/license.txt |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE - Universidade Estadual do Oeste do Paraná (UNIOESTE) |
repository.mail.fl_str_mv |
biblioteca.repositorio@unioeste.br |
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1811723393138950144 |