Construction of mutant Trichoderma reesei strains and analysis of regulatory elements aiming to identify and characterize new genes with biotechnological potential for lignocellulose deconstruction

• Grant number: 17/18987-7
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: November 30, 2017
• End date: June 03, 2018
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Juliana Velasco de Castro Oliveira
• Grantee: Gustavo Pagotto Borin
• Supervisor: Astrid Mach-Aigner
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Institution abroad: Vienna University of Technology (TU Wien), Austria
• Associated to the scholarship: 15/08222-8 - Analysis of transcriptional co-regulation and identification of genes of biotechnological interest in Trichoderma reesei, BP.DR

Abstract

In Brazil, sugarcane bagasse has been proposed as a promising residue for second-generation (2G)ethanol production due to its abundance and low cost. However, biotechnological challenges mustbe overcome to make this fuel an economically viable substitute for fossil fuels. In this regard, it isnecessary to reduce the cost of enzyme mixtures used to break down the lignocellulose and releasethe fermentable sugars. The biomass-degrading fungus Trichoderma reesei has been considered amodel for cellulose degradation and its enzymes have been used in several cocktails. In spite of allgenetic efforts that have been made, it is not completely clear how this fungus uses its cellulolyticsystem to deconstruct the plant cell wall neither the fine-tuning regulatory elements involved in thisprocess. Recently, the transcriptome of T. reesei RUT C-30 grown on steam-exploded sugarcanebagasse was investigated and several differentially expressed genes (DEGs) were found up-regulatedin this carbon source, including CAZymes, sugar transporters, transcription factors anduncharacterized genes coding for proteins with signal peptide. From the RNA-Seq data, a gene coexpression network analysis was carried out to identify DEGs that were co-expressed in bagasse andmight be related to the lignocellulosic cell wall deconstruction. Next, an in silico analysis wasperformed to search for XYR1 and CRE1 regulatory motifs at the promoter region of the genespresent within the network. As part of the student's doctorate, five genes were chosen to be deletedor overexpressed in T. reesei aiming to elucidate their function in lignocellulose breakdown.However, despite several attempts, the student did not have success to transform T. reesei. Thistechnique has proved not easy to be performed and it depends on various factors, such as theselected strain, protocol and protoplast formation. Thus, one of the main aims of this proposal is tolearn and carry out T. reesei transformation at Dr. Astrid Mach-Aigner's research group. This grouphas great expertise with genetic engineering of T. reesei and has contributed deeply to its biologyknowledge, especially in relation to the transcriptional regulation of CAZymes during lignocellulosedegradation. In addition, it is proposed to evaluate in vivo the influence of chromatin packaging tothe regulation of genes having XYR1/CRE1 motif sites at the promotor region under inducing(cellulose and lactose) and repressing (glucose) conditions through the CHART-PCR. This metodologyis interesting to detect the accessibility of chromatin and it is routinely carried out at Dr. MachAigner's group. At the end of this internship, the knowledge acquired will be useful to study newgenes potentially involved in bagasse cell wall deconstruction. The findings can help to improve theenzymatic cocktails, resulting in better perspectives to 2G ethanol economic viability.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

BONIN, GUSTAVO PAGOTTO; CARAZZOLLE, MARCELO FALSARELLA; CORREA DOS SANTOS, RENATO AUGUSTO; RIANO-PACHON, DIEGO MAURICIO; DE CASTRO OLIVEIRA, JULIANA VELASCO. Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 6, OCT 22 2018. (14/11766-7, 17/18987-7, 15/08222-8, 14/15799-7)

BORIN, GUSTAVO PAGOTTO; OLIVEIRA, JULIANA VELASCO DE CASTRO. Assessing the intracellular primary metabolic profile of Trichoderma reesei and Aspergillus niger grown on different carbon sources. FRONTIERS IN FUNGAL BIOLOGY, v. 3, p. 20-pg., 2022-09-27. (15/08222-8, 17/18987-7, 22/00474-1)