Advanced search
Start date
Betweenand


Genetic manipulation of differentially expressed transcription factors under induction of expression of heterologous enzymes in Aspergillus nidulans

Full text
Author(s):
Everton Paschoal Antoniel
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
André Ricardo de Lima Damásio; Rafael Silva Rocha; Fernando Segato
Advisor: André Ricardo de Lima Damásio
Abstract

As a way to lessen dependence on fossil fuels, biofuels and chemical products obtained through enzymatic hydrolysis of plant biomass, has been the focus of several scientific investigations. However, the recalcitrance of the plant cell wall, and the high price of hydrolytic enzymes mean that its complete and efficient degradation is still a challenge to be overcome for its economic viability. Therefore, the main goal of this project is to evaluate the influence of genetic manipulation of transcription factors (TFs) on the levels of production/secretion of degrading enzymes from plant biomass. It is known that there is a direct correlation between overexpression of genes and endoplasmic reticulum stress in filamentous fungi. In response to this, a cell triggers an intense program, called unfolded protein response, to maintain cellular proteostasis. Thus, the TFs manipulated in this work were selected from RNAseq data, enrichment of GO terms and genetic co-expression network of the fungus model Aspergillus nidulans exposed to cell stress induced by overexpression and secretion of heterologous proteins. This analysis resulted in the selection of 5 TFs - AN8772, AN9373, AN7331, AN7913 and AN7592 - which were in co-expression modules enriched with GO terms such as: proteasomal ubiquitin-independent protein catabolic process (GO: 0010499), sporulation (GO: 0043934), calcium ion homeostasis (GO: 0055074), Golgi vesicle transport (GO: 0048193), translation (GO: 0006412), protein refolding (GO: 0042026) and small molecule catabolic process (GO: 0044282). The 5 TFs were successfully deleted in A. nidulans using the CRISPR-Cas9 technique. The phenotype of the mutant strains was evaluated for cell wall integrity (Congo Red - CR and Calcofluor White - CFW), osmotic stress (Sorbitol), ionic stress (CaCl2), oxidative stress (Menadione), sporulation and protease secretion. The strain delta AN7913 and delta AN7592 were the most resistant to stressors of cell wall integrity. The strain delta AN7592 was the only one to present reduced sporulation and resistance to osmotic stress. To evaluate the oxidative stress response, the strain delta AN8772 was more sensitive, while delta AN7913 was more resistant. In the protease analysis, the strains delta AN9373, delta AN7331 and delta AN7913 had an increase in the secretion of proteases, demonstrating that these TFs may have a potential function in the regulation of the secretion pathway. In addition, the process of constructing strains overexpressing a heterologous enzyme is underway and will be able to answer whether these TFs have an influence on the secretion of heterologous enzymes in A. nidulans. It was concluded that it was possible to select TFs potentially involved in the protein secretion pathway in A. nidulans using transcriptome data and coexpression networks. In addition, we demonstrated that the deleted TFs are involved in different biological processes, highlighting our preliminary data that demonstrate greater secretion of proteases in some mutants. Finally, we intend to deepen our studies by carrying out additional experiments to assess the secretion levels of these mutants through the overexpression of a target recombinant enzyme and, eventually, to describe the mechanism that induces this possible increase in secretion (AU)

FAPESP's process: 17/26315-9 - Influence of transcription factors on the secretion of enzymes in Aspergillus nidulans
Grantee:Everton Paschoal Antoniel
Support Opportunities: Scholarships in Brazil - Master