Application of a guide RNA library based on the dCas9 system towards the improvement of xylose consumption in S. cerevisiae

• Grant number: 21/02421-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: December 01, 2021
• End date: April 30, 2023
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Gonçalo Amarante Guimarães Pereira
• Grantee: Frank Uriel Suarez Lizarazo
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Despite countless efforts to make hemicellulosic bioethanol production feasible, the metabolism of pentoses by the yeast Saccharomyces cerevisiae remains as one of the limiting factors in the process. Metabolic engineering to improve this phenotype is the focus of research projects by different groups, almost always limited to traditional regulation based on strong overexpression or deletion of target genes. Even though the contribution of classical genetics of allele deletion/insertion has proven to be quite effective, there remains a need and importance to generate new results through assays that allow the incorporation of moderate levels of expression to identify new targets that could increase the process efficiency. Using the strain LVY34.4, PE-2 derived, modified and evolved for xylose consumption, a methodology based on sgRNA libraries and the CRISPR-dCas9 system will be implemented for a moderate regulation of gene expression. dCas9-VPR vectors will be responsible for activating the expression, while dCas9-Mxi1 vectors will act in suppressing the transcription of up to 969 genes participating in metabolic pathways. From xylose growth assays, targets will be identified and what level of regulation allows for better metabolic and fermentative capacity.