Transcriptional co-regulation analysis and identification of genes of biotechnological interest in Trichoderma reesei

Author(s)

Gustavo Pagotto Borin

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-08-27

Examining board members

Juliana Velasco de Castro Oliveira; Anete Pereira de Souza; Rafael Silva Rocha; Richard John Ward; Marcelo Mendes Brandão

• Advisor: Juliana Velasco de Castro Oliveira

Abstract

Biofuels, especially second generation (2G) ethanol, are today one of the main sustainable alternatives to the replacement of fossil fuels towards the consolidation of a circular bioeconomy. In Brazil, 2G ethanol is produced from bagasse and sugarcane straw, which are lignocellulosic residues generated in abundance in the sugar-energy industry. This technology, however, still presents some economic limitations, such as the high cost of the enzymatic cocktails necessary for the hydrolysis of lignocellulosic biomass. The filamentous fungus Trichoderma reesei is considered the main biological platform of the production and secretion of cellulases and hemicellulases that compose enzymatic cocktails. Although the cellulolytic system and transcriptional regulation of T. reesei are well studied, efforts are still needed to investigate the entire set of genes related to the degradation of lignocellulosic biomass by this fungus. Thus, the main objectives of this study are the inference of a gene co-expression network based on the T. reesei RUT-C30 transcriptome and the characterization of a few genes with biotechnological potential in mutant knockout strains. The co-expression network analysis separated the upregulated genes in bagasse (BEX) into several modules, according to the gene expression profile. Several genes encoding (hemi)cellulases, sugar transporters, transcription factors and hypothetical proteins were coexpressed in the same modules. Central genes from these modules (hubs) were identified, and included transcription factors and hypothetical proteins still not characterized. The in silico prediction of DNA binding sites in the promoter region for XYR1, the major cellulase activator, has also revealed interesting genes that can be regulated by this transcription factor. From the network analysis, six genes were chosen to be deleted and characterized in T. reesei. Four of them were deleted successfully and one gene encoding a hypothetical zinc finger transcription factor was shown to play a positive role in bagasse degradation, since its deletion significantly decreased the activities of cellobiohydrolase (pNPC) and beta-glucosidase (pNPG) at this carbon source. Gene expression data by qPCR have also demonstrated that this gene is targeted by the CRE1-mediated catabolite repression. In addition, the metabolic profile of T. reesei and Aspergillus niger grown on glucose, soluble cellulose (CMC), lactose and pretreated bagasse was investigated by NMR to evaluate the metabolic pathways employed by these two industrial fungi. The carbon source used was the major responsible for the data variation, suggesting that both fungi adopt similar strategies to grown and adapt to the substrates evaluated, even they had evolved in an independent way. Metabolites of energy reserves (mannitol and trehalose), phospholipids (glycerol-3-phosphocholine) and amino acid metabolites (glutamine, alanine and glutamate) were significantly more abundant in all conditions for both fungi. T. reesei assimilated lactose more efficiently, and it seems to activate the TCA alternative cycle (glyoxalate shunt) in CMC as strategy to produce energy by gluconeogenese. On the other hand, A. niger showed higher levels of glycerol and GABA in CMC and lactose. In BEX, A. niger had greater cellulase and hemicellulase activity, contributing to the activation of glycolysis. As conclusions of this work, the gene co-expression network analysis allowed the identification of a large number of genes with biotechnological potential in the degradation of sugarcane bagasse. The characterization of the knockout mutants led to the discovery of a putative transcriptional activator of cellulases, but additional studies must be conducted to better understand its role in T. reesei gene regulation. The metabolic profile of T. reesei and A. niger had also revealed that both fungi use similar strategies with a few differences in the assimilation of different carbon sources, and contributed to a better understanding of fungal physiology under conditions that induce the production of hydrolytic enzymes. Finally, the results obtained in this work contributed to a better understanding of the T. reesei cellulolytic system and may help to reduce the costs associated with 2G ethanol (AU)

• FAPESP's process: 15/08222-8 - Analysis of transcriptional co-regulation and identification of genes of biotechnological interest in Trichoderma reesei
• Grantee: Gustavo Pagotto Borin
• Support Opportunities: Scholarships in Brazil - Doctorate