Identification and characterization of a new transcription factor, homologous to Saccharomyces cerevisiae Azf1, involved in the regulation of cellulase expression in the filamentous fungus Trichoderma reesei

Author(s)

Amanda Cristina Campos Antoniêto

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2018-05-09

Examining board members

Roberto do Nascimento Silva; Gilberto Ubida Leite Braga; Fernanda Zanolli Freitas; María Eugenia Guazzaroni; Richard John Ward

• Advisor: Roberto do Nascimento Silva

Abstract

The fungus Trichoderma reesei is known for its high capacity in secrete cellulolytic enzymes that act in the degradation of the cellulose polymer into glucose molecules, being a key point in the production of second generation ethanol. The present work aims to contribute to the understanding of the molecular mechanisms involved in the process of biomass deconstruction in T. reesei through the identification of new transcription factors associated to this process. For this, we screened 14 transcription factors potentially involved in the degradation of biomass and identified the gene Tr103275, homologous to the Azf1 regulator of Saccharomyces cerevisiae. Posteriorly, a mutant strain with deletion of azf1 was obtained. During growth in cellulose and sugarcane bagasse, azf1 was highly induced in T. reesei, while glucose repressed the gene expression of this transcription factor. The absence of azf1 affected growth and sporulation in T. reesei, since the mutant strain produced less and smaller spores relative to the parental strain. In addition, we have shown that TrAzf1 acts as a positive regulator of the expression of holocellulolytic enzymes, whereas the genes coding for CBHs, EGs, BGs and xyn4 were down-regulated in the Δazf1 mutant in cellulose and sugarcane bagasse. The binding site for TrAzf1 was predicted in this work through bioinformatics analysis. To evaluate the interaction of TrAzf1 with this DNA binding site, the recombinant Azf1 zinc finger motif was obtained by heterologous expression in E. coli and used in the SPR analyzes. However, validation of the transcription factor-DNA interaction by SPR was not conclusive and other approaches should be performed to validate the binding motif of TrAzf1 in T. reesei. The role of Azf1 in regulating the degradation of plant biomass was described for the first time in this work. This transcription factor is a potential target for the genetic manipulation of T. reesei, aiming to improve the production of holocellulolytic enzymes by this species. (AU)

• FAPESP's process: 14/09493-2 - Identification and characterization of novel transcription factors involved in the regulation of lignocellulosic biomass degradation in Trichoderma reesei (Hypocrea jecorina)
• Grantee: Amanda Cristina Campos Antoniêto
• Support Opportunities: Scholarships in Brazil - Doctorate