Comparative analysis of the heterologous protein secretion in Aspergillus nidulans

Author(s)

Felipe Calzado

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2017-03-13

Examining board members

André Ricardo de Lima Damásio; Roberto do Nascimento Silva; Flavia Fisch Winck

• Advisor: Gabriela Felix Persinoti; André Ricardo de Lima Damásio

Abstract

As a way to reduce the use of fossil fuels and increase the use of renewable energies, there is a major recent worldwide incentive in the development of biorefineries, which involves integrated conversion processes of plant biomass and equipment for the production of energy, fuels and renewable chemicals. In this perspective, the second generation of ethanol obtained from the conversion of lignocellulosic biomass to fermentable sugars has been the focus of many currently researches in order to make its use feasible. However, due to the recalcitrant nature of lignocellulosic biomass, the current processing cost in the production of biofuels becomes an important bottleneck to be overcome. One of the ways to overcome this problem is to improve the production of enzymes required in the enzymatic hydrolysis of plant biomass, a process still considered very expensive nowadays. The filamentous fungi stand out in the production and secretion of lignocellulolytic enzymes in a homologous and heterologous way, however little is known about the regulation of the protein secretion mechanisms in these microorganisms. Considering this, the use of the filamentous fungus model Aspergillus nidulans becomes an important resource of studies to be well genetically characterized. Our objective in this work was to perform a comparative analysis of the mechanisms of secretion of different heterologous proteins in A. nidulans through transcriptome data analysis using RNA-seq technique (Next Generation RNA sequencing). To monitor the secretion process, we adopted 3 target genes: alfa-arabinofuranosidase GH51 (AbfA) and beta-glucosidase GH3 (BglC) from A. fumigatus, and thermophilic mannanase GH5 (1542) from Thermotoga petrophila. These genes were transformed into A. nidulans, and for AbfA high secretion levels were reached, different from BglC and 1542, where secretion levels were minimal. Thereby, transcriptomic profiles of the genes related to protein secretion were defined in A. nidulans, representing the super expression response of three heterologous genes at different induction times. In general there were overrepresented biological processes related mainly to metabolism, protein with binding function, cellular transport and defense. In addition, 17 differentially expressed genes directly involved with the A. nidulans secretion pathway were identified, representing important targets for the genetic manipulation of this organism. This was the first study to describe the transcriptomic response of heterologous protein secretion in A. nidulans, generating a new perspective when comparing three different heterologous proteins with increasing levels of secretion (AU)

• FAPESP's process: 14/23051-2 - Comparative analysis of the heterologous protein secretion mechanisms in Aspergillus nidulans
• Grantee: Felipe Calzado
• Support Opportunities: Scholarships in Brazil - Master