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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2022.tde-09052022-101527
Document
Author
Full name
Alinne Costa Silva
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2021
Supervisor
Committee
Silva, Roberto do Nascimento (President)
Noronha, Eliane Ferreira
Ruller, Roberto
Ward, Richard John
Title in Portuguese
Análise global da regulação transcricional do gene de cerato-platanina epl1 em Trichoderma reesei
Keywords in Portuguese
Cerato-platanina
epl1
RNA-seq
Trichoderma reesei
Abstract in Portuguese
Trichoderma reesei é um dos fungos mais bem estudados e um dos principais produtores de celulases, enzimas que atuam na degradação de biomassa lignocelulósica. Apesar do amplo conhecimento sobre o sistema celulolítico desse fungo, pouco se sabe sobre a forma como ele reconhece o meio extracelular. Nesse contexto, a família de proteínas cerato-plataninas (CPs) tem ganhado destaque por sua capacidade de se ligar e afrouxar resíduos celulósicos. CPs são encontradas exclusivamente em fungos e apresentam similaridade estrutural com expansinas vegetais. Este trabalho tem como objetivo contribuir para o entendimento do papel de EPL1 de T. reesei. Para isso, foi feita a construção do mutante nocaute (Δepl1) e os experimentos foram realizados em comparação com o parental QM6a. A deleção de epl1 não alterou as atividades celulolíticas do fungo quando cultivado em celulose em estado submerso (FS), no entanto, quando cultivado em estado semi-sólido (FES) com bagaço de cana de açúcar, houve diminuição da atividade β-glicosidase. Para a análise do transcriptoma global de Δepl1, foi realizado o sequenciamento de nova geração das linhagens mutante e parental cultivadas em celulose 1% por 72 horas. Dos 9.143 genes anotados no genoma de T. reesei, 760 foram considerados diferencialmente expressos, sendo 260 exclusivos para QM6a, 214 genes exclusivos em Δepl1 e 286 compartilhado entre eles. A maioria dos genes que estavam up regulados no mutante pertenciam a categorias do Gene Ontology descritas como transporte de ácido orgânico, atividade oxidorredutase, transporte, ligação de polissacarídeo e ligação de carboidratos. As Enzimas ativas de Carboidratos (CAZymes) envolvidas no remodelamento da parede do fungo mostrou-se up reguladas na linhagem Δepl1. Os resultados mostram o papel de EPL1 no reconhecimento e fisiologia do fungo durante a produção de celulases. Espera-se com esses resultados contribuir para uma melhor compreensão do papel desempenhando por EPL1 em T. reesei.
Title in English
Global analysis of transcriptional regulation of the cerato-platanin gene epl1 in Trichoderma reesei
Keywords in English
Cerato-platanin
epl1
RNA-seq
Trichoderma reesei
Abstract in English
Trichoderma reesei is one of the most studied fungi and one of the main producers of cellulases, enzymes that hydrolyse lignocellulosic biomass. Despite the extensive knowledge about its cellulolytic set of enzymes, little is known about how T. reesei recognizes the extracellular environment. In this context, the family of cerato-platanin proteins (CPs) found exclusively in fungi and showing structural similarity to plant expansins - has been gaining prominence for possessing binding and loosening on the cellulose structure. In this work, we used transcriptomic analysis to describe the role of EPL1, a cerato-platanin from T. reesei, on cellulose growth. For this, we constructed the T. reesei Δepl1 mutant, which didn't show difference in the enzymatic activity when grown on cellulose, however, when grown in FES media supplemented with sugarcane bagasse, it showed a decrease in β-glycosidase activity when compared with the parental QM6a strain. We performed the global transcriptome analysis - through next generation sequencing - of the Δepl1 mutant and the parental QM6a strains after grown in 1% cellulose for 72 hours. From the 9,143 genes annotated in the T. reesei genome, 760 were found to be differentially expressed between Δepl1 and QM6a; being 260 genes unique to QM6a, 214 unique to Δepl1, and 286 commonly shared between them. Most of the genes that were up regulated in the mutant belonged to Gene Ontology categories described as organic acid transport, oxidoreductase activity, transport, polysaccharide binding, and carbohydrate binding. Active Carbohydrate Enzymes (CAZymes) involved in fungal wall remodeling were shown to be upregulated in the epl1 strain. The results show the role of EPL1 in the recognition and physiology of the fungus during cellulase production. These results are expected to contribute to a better understanding of the role played by EPL1 in T. reesei.
 
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ALINNECOSTASILVA.pdf (3.27 Mbytes)
Publishing Date
2022-05-10
 
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