Gene editing of the model yeast Pichia pastoris using the CRISPR-Cas system and transformation of the enzyme alternative oxidase from the fungus Ceratocystis cacaofunesta, causal agent of the wilt of cocoa

• Grant number: 16/24264-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2017
• End date: March 31, 2018
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Gonçalo Amarante Guimarães Pereira
• Grantee: Laura Beatrice Poll
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

The cocoa tree (Theobroma cacao) is of great economic importance to Brazil, since it produces cocoa beans that are used to make widely appreciated chocolate. Fungal diseases are one of the major factors affecting the world cocoa production. In Brazil, one of the main pathogens is the ascomycete Ceratocystis cacaofunesta, which causes the wilt of cocoa. Efforts aimed to better understand C. cacaofunesta's biology led to the identification of the mitochondrial enzyme Alternative Oxidase (CcAOX), a key component for this fungu's development. Therefore, CcAOX is a potential molecular target to control the wilt of cocoa, as well as other phytopathogens present in Brazil that depended on the activity of this enzyme for growth or survival. Our group has planned and synthetized new AOX inhibitors that were evaluated on the yeast Pichia pastoris. However, in order to understand with more clarity the potential antifungal activity of these molecules against C. cacaofunesta, it is crucial to assess their effect on CcAOX. Therefore, this project aims to genetically manipulate P. pastoris, specifically to knockdown its own AOX and to transform CcAOX. With this, it will be possible to take advantage of methodologies that were already developed with P. pastoris, which, in turn, would aid in the search of new antifungal agents. (AU)