Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis
Ano de defesa: | 2009 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , , |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Viçosa
|
Programa de Pós-Graduação: |
Mestrado em Microbiologia Agrícola
|
Departamento: |
Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
|
País: |
BR
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://locus.ufv.br/handle/123456789/5307 |
Resumo: | This work is a contribution to the studies about the role of KlRox1p in fermentative metabolism of Kluyveromyces lactis. Rox1p is known in Saccharomyces cerevisiae by repressing in aerobic conditions genes that are expressed when oxygen is limited. The kinetics growth of lineages of Kluyveromyces lactis MW270-7B and its derivative mutant K. lactis MW270- 7B rox1Δ was carry out on YNB media (Yeast Nitrogen Base) containing fermentative carbon sources (glucose and lactose) or non fermentative carbon sources (glycerol and ethanol) to determine the growth profile on this sources. Besides both yeasts had its fermentative capacity researched in the presence of an inhibitor of electron chain carrier, antimicin A on YPD (yeast extract, peptone and glucose). The results indicated that the absence of KlROX1 didn't favor the growth in fermentative carbon sources, but it favored in non fermentative carbon sources. The more fermentative capacity of K. lactis rox1Δ was confirmed for the higher ethanol yield for biomass detected in the absence and presence of antimicin A (10 and 20 µM). The lineages rox1 mutant and control were also cultivated on YNB plus glucose under aerobic continuous culture (D = 0,01 h-1) and later they were submitted to a hypoxic condition for 9 hours. Samples were collected every 3 hours for analysis of the expression of putative target genes of KlRox1p as well as analysis of ethanol and of the enzymatic activity of the alcohol desidrogenase (ADH). For the analysis of gene expression, recognition sequences for KlRox1p were researched in silico in the promoters of the genes KlAAC3, KlCOX5B, KlHEM13, KlHAP1, KlRAG1 and KlADH1 and they were found diverging of two nucleotide of the consensus sequence YYYATTGTTCTC. KlRox1p showed to have negative effect on expression of KlAAC3 and KlHEM13, but it didn't seem to act on KlCOX5B. The ethanol concentration, the activity of ADH and the expression of KlADH1 evidenced the metabolism more fermentative of K. lactis rox1Δ on aerobic and hypoxic conditions. And in a way not known, KlRox1p acts on expression of KlHAP1 and KlRAG1. The influence of KlRox1p on expression of KlADH1, KlRAG1 and KlHAP1 suggests the participation of KlRox1p in the control of key points of the fermentative metabolism: entrance of the glucose that determine the flow for the maintenance of fermentative way, as well as in the reductive reaction end that culminates with the ethanol production. The existence of genes that showed to be regulated negatively by KlRox1p in aerobic condition, like KlAAC3 and KlHEM13, indicates the existence in K. lactis of mechanism of transcriptional regulation for oxygen that intends to better use the oxygen in a limit condition, similar to the function of Rox1p in Saccharomyces cerevisiae. However that mechanism seems not just to be related with the concentrations of oxygen, but also with the carbon sources used, fermentative sources or non fermentative sources. |
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Harami, Talitahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4750606T5Fietto, Luciano Gomeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8Mantovani, Hilário Cuquettohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026Z7Passos, Flávia Maria Lopeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781817D3Bazzolli, Denise Mara Soareshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4761710D6Vanetti, Maria Cristina Dantashttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783874H3Ramos, Juliana Rocha Lopes Soareshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4790613J32015-03-26T13:51:46Z2009-08-032015-03-26T13:51:46Z2009-02-16HARAMI, Talita. Influence of KlRox1p on fermentative metabolism of Kluyveromyces lactis. 2009. 62 f. Dissertação (Mestrado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2009.http://locus.ufv.br/handle/123456789/5307This work is a contribution to the studies about the role of KlRox1p in fermentative metabolism of Kluyveromyces lactis. Rox1p is known in Saccharomyces cerevisiae by repressing in aerobic conditions genes that are expressed when oxygen is limited. The kinetics growth of lineages of Kluyveromyces lactis MW270-7B and its derivative mutant K. lactis MW270- 7B rox1Δ was carry out on YNB media (Yeast Nitrogen Base) containing fermentative carbon sources (glucose and lactose) or non fermentative carbon sources (glycerol and ethanol) to determine the growth profile on this sources. Besides both yeasts had its fermentative capacity researched in the presence of an inhibitor of electron chain carrier, antimicin A on YPD (yeast extract, peptone and glucose). The results indicated that the absence of KlROX1 didn't favor the growth in fermentative carbon sources, but it favored in non fermentative carbon sources. The more fermentative capacity of K. lactis rox1Δ was confirmed for the higher ethanol yield for biomass detected in the absence and presence of antimicin A (10 and 20 µM). The lineages rox1 mutant and control were also cultivated on YNB plus glucose under aerobic continuous culture (D = 0,01 h-1) and later they were submitted to a hypoxic condition for 9 hours. Samples were collected every 3 hours for analysis of the expression of putative target genes of KlRox1p as well as analysis of ethanol and of the enzymatic activity of the alcohol desidrogenase (ADH). For the analysis of gene expression, recognition sequences for KlRox1p were researched in silico in the promoters of the genes KlAAC3, KlCOX5B, KlHEM13, KlHAP1, KlRAG1 and KlADH1 and they were found diverging of two nucleotide of the consensus sequence YYYATTGTTCTC. KlRox1p showed to have negative effect on expression of KlAAC3 and KlHEM13, but it didn't seem to act on KlCOX5B. The ethanol concentration, the activity of ADH and the expression of KlADH1 evidenced the metabolism more fermentative of K. lactis rox1Δ on aerobic and hypoxic conditions. And in a way not known, KlRox1p acts on expression of KlHAP1 and KlRAG1. The influence of KlRox1p on expression of KlADH1, KlRAG1 and KlHAP1 suggests the participation of KlRox1p in the control of key points of the fermentative metabolism: entrance of the glucose that determine the flow for the maintenance of fermentative way, as well as in the reductive reaction end that culminates with the ethanol production. The existence of genes that showed to be regulated negatively by KlRox1p in aerobic condition, like KlAAC3 and KlHEM13, indicates the existence in K. lactis of mechanism of transcriptional regulation for oxygen that intends to better use the oxygen in a limit condition, similar to the function of Rox1p in Saccharomyces cerevisiae. However that mechanism seems not just to be related with the concentrations of oxygen, but also with the carbon sources used, fermentative sources or non fermentative sources.Esse trabalho é uma contribuição aos estudos sobre a função de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis. Rox1p é conhecida em Saccharomyces cerevisiae por reprimir, em aerobiose, genes que são expressos em condição limitante de oxigênio. A cinética de crescimento de linhagens de Kluyveromyces lactis MW270-7B e sua mutante derivativa K. lactis MW270-7B rox1Δ foi realizada em meio YNB (Yeast Nitrogen Base) contendo fontes de carbono fermentáveis (glicose e lactose) ou não fermentáveis (glicerol e etanol) para determinar o perfil de crescimento nessas fontes. Além disso, ambas as linhagens tiveram sua capacidade fermentativa avaliada na presença de um inibidor da cadeia transportadora de elétrons, antimicina A, em meio YPD (extrato de levedura, peptona e glicose). Os resultados indicaram que a ausência de KlROX1 não favoreceu o crescimento em fontes de carbono fermentáveis, mas favoreceu em fontes de carbono não fermentáveis. A maior capacidade fermentativa de K. lactis rox1Δ foi confirmada pelo maior rendimento de etanol por biomassa detectada, na ausência e presença de antimicina A (10 e 20 µM). As linhagens mutante rox1 e controle também foram cultivadas em YNB e glicose em regime contínuo (D= 0,1h-1) sob aerobiose e, em seguida, submetidas à hipoxia por 9 horas. Amostras foram coletadas a cada 3 horas para análise da expressão de possíveis genes alvos de KlRox1p, bem como de etanol e da atividade da álcool desidrogenase (ADH). Para análise da expressão gênica, sequências de reconhecimento por KlRox1p foram pesquisadas in silico nos promotores dos genes KlAAC3, KlCOX5B, KlHEM13, KlHAP1, KlRAG1 e KlADH1 e foram encontradas divergindo de dois nucleotídeos da sequência consenso YYYATTGTTCTC. KlRox1p mostrou ter efeito negativo sobre as expressões de KlAAC3 e KlHEM13 em aerobiose, mas não pareceu atuar sobre KlCOX5B. A concentração de etanol, a atividade da ADH e a expressão de KlADH1 evidenciaram o metabolismo mais fermentativo de K. lactis rox1Δ tanto em aerobiose quanto em hipoxia. E, por um mecanismo ainda desconhecido, KlRox1p atuou sobre as expressões de KlHAP1 e KlRAG1. A influência de KlRox1p na expressão de KlADH1, KlRAG1 e KlHAP1, sugere a participação de KlRox1p no controle de pontos chaves do metabolismo fermentativo: entrada da glicose, que determina o fluxo para a manutenção de uma via fermentativa, bem como na reação redutiva final, que culmina com a formação de etanol. A existência de genes que mostraram ser regulados negativamente por KlRox1p em aerobiose, como KlAAC3 e KlHEM13, indica a existência em K. lactis de mecanismo de regulação transcricional por oxigênio que visa melhor utilizar o oxigênio numa condição limitante, semelhante à função de Rox1p em Saccharomyces cerevisiae. Esse mecanismo, entretanto, parece não estar relacionado apenas com as concentrações de oxigênio, mas também com metabolismo de carbono fermentável ou não fermentável.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Microbiologia AgrícolaUFVBRAssociações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesseKluyveromyces lactisMetabolismo fermentativoExpressão gênicaKluyveromyces lactisFermentative metabolismGene expressionCNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOSInfluência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactisInfluence of KlRox1p on fermentative metabolism of Kluyveromyces lactisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf697140https://locus.ufv.br//bitstream/123456789/5307/1/texto%20completo.pdfa673ff7affad18149751f45b84127b29MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain88748https://locus.ufv.br//bitstream/123456789/5307/2/texto%20completo.pdf.txt478e70351b8f797d3276a941ecba15f2MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3545https://locus.ufv.br//bitstream/123456789/5307/3/texto%20completo.pdf.jpgb970b55c67c2416d2e5091fc6652958fMD53123456789/53072016-04-10 23:16:20.942oai:locus.ufv.br:123456789/5307Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-11T02:16:20LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
dc.title.alternative.eng.fl_str_mv |
Influence of KlRox1p on fermentative metabolism of Kluyveromyces lactis |
title |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
spellingShingle |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis Harami, Talita Kluyveromyces lactis Metabolismo fermentativo Expressão gênica Kluyveromyces lactis Fermentative metabolism Gene expression CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS |
title_short |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
title_full |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
title_fullStr |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
title_full_unstemmed |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
title_sort |
Influência de KlRox1p no metabolismo fermentativo de Kluyveromyces lactis |
author |
Harami, Talita |
author_facet |
Harami, Talita |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4750606T5 |
dc.contributor.author.fl_str_mv |
Harami, Talita |
dc.contributor.advisor-co1.fl_str_mv |
Fietto, Luciano Gomes |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8 |
dc.contributor.advisor-co2.fl_str_mv |
Mantovani, Hilário Cuquetto |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026Z7 |
dc.contributor.advisor1.fl_str_mv |
Passos, Flávia Maria Lopes |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781817D3 |
dc.contributor.referee1.fl_str_mv |
Bazzolli, Denise Mara Soares |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4761710D6 |
dc.contributor.referee2.fl_str_mv |
Vanetti, Maria Cristina Dantas |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783874H3 |
dc.contributor.referee3.fl_str_mv |
Ramos, Juliana Rocha Lopes Soares |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4790613J3 |
contributor_str_mv |
Fietto, Luciano Gomes Mantovani, Hilário Cuquetto Passos, Flávia Maria Lopes Bazzolli, Denise Mara Soares Vanetti, Maria Cristina Dantas Ramos, Juliana Rocha Lopes Soares |
dc.subject.por.fl_str_mv |
Kluyveromyces lactis Metabolismo fermentativo Expressão gênica |
topic |
Kluyveromyces lactis Metabolismo fermentativo Expressão gênica Kluyveromyces lactis Fermentative metabolism Gene expression CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS |
dc.subject.eng.fl_str_mv |
Kluyveromyces lactis Fermentative metabolism Gene expression |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS |
description |
This work is a contribution to the studies about the role of KlRox1p in fermentative metabolism of Kluyveromyces lactis. Rox1p is known in Saccharomyces cerevisiae by repressing in aerobic conditions genes that are expressed when oxygen is limited. The kinetics growth of lineages of Kluyveromyces lactis MW270-7B and its derivative mutant K. lactis MW270- 7B rox1Δ was carry out on YNB media (Yeast Nitrogen Base) containing fermentative carbon sources (glucose and lactose) or non fermentative carbon sources (glycerol and ethanol) to determine the growth profile on this sources. Besides both yeasts had its fermentative capacity researched in the presence of an inhibitor of electron chain carrier, antimicin A on YPD (yeast extract, peptone and glucose). The results indicated that the absence of KlROX1 didn't favor the growth in fermentative carbon sources, but it favored in non fermentative carbon sources. The more fermentative capacity of K. lactis rox1Δ was confirmed for the higher ethanol yield for biomass detected in the absence and presence of antimicin A (10 and 20 µM). The lineages rox1 mutant and control were also cultivated on YNB plus glucose under aerobic continuous culture (D = 0,01 h-1) and later they were submitted to a hypoxic condition for 9 hours. Samples were collected every 3 hours for analysis of the expression of putative target genes of KlRox1p as well as analysis of ethanol and of the enzymatic activity of the alcohol desidrogenase (ADH). For the analysis of gene expression, recognition sequences for KlRox1p were researched in silico in the promoters of the genes KlAAC3, KlCOX5B, KlHEM13, KlHAP1, KlRAG1 and KlADH1 and they were found diverging of two nucleotide of the consensus sequence YYYATTGTTCTC. KlRox1p showed to have negative effect on expression of KlAAC3 and KlHEM13, but it didn't seem to act on KlCOX5B. The ethanol concentration, the activity of ADH and the expression of KlADH1 evidenced the metabolism more fermentative of K. lactis rox1Δ on aerobic and hypoxic conditions. And in a way not known, KlRox1p acts on expression of KlHAP1 and KlRAG1. The influence of KlRox1p on expression of KlADH1, KlRAG1 and KlHAP1 suggests the participation of KlRox1p in the control of key points of the fermentative metabolism: entrance of the glucose that determine the flow for the maintenance of fermentative way, as well as in the reductive reaction end that culminates with the ethanol production. The existence of genes that showed to be regulated negatively by KlRox1p in aerobic condition, like KlAAC3 and KlHEM13, indicates the existence in K. lactis of mechanism of transcriptional regulation for oxygen that intends to better use the oxygen in a limit condition, similar to the function of Rox1p in Saccharomyces cerevisiae. However that mechanism seems not just to be related with the concentrations of oxygen, but also with the carbon sources used, fermentative sources or non fermentative sources. |
publishDate |
2009 |
dc.date.available.fl_str_mv |
2009-08-03 2015-03-26T13:51:46Z |
dc.date.issued.fl_str_mv |
2009-02-16 |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:51:46Z |
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 |
HARAMI, Talita. Influence of KlRox1p on fermentative metabolism of Kluyveromyces lactis. 2009. 62 f. Dissertação (Mestrado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2009. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/5307 |
identifier_str_mv |
HARAMI, Talita. Influence of KlRox1p on fermentative metabolism of Kluyveromyces lactis. 2009. 62 f. Dissertação (Mestrado em Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse) - Universidade Federal de Viçosa, Viçosa, 2009. |
url |
http://locus.ufv.br/handle/123456789/5307 |
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Universidade Federal de Viçosa |
dc.publisher.program.fl_str_mv |
Mestrado em Microbiologia Agrícola |
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UFV |
dc.publisher.country.fl_str_mv |
BR |
dc.publisher.department.fl_str_mv |
Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse |
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Universidade Federal de Viçosa |
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