Protease deletion in Aspergillus oryzae as a strategy to optimize heterologous protein production

• Grant number: 25/06509-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: January 01, 2026
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: André Ricardo de Lima Damasio
• Grantee: Guilherme Guimarães Ortolan
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 22/05731-2 - BEYOND: establishing a fungal cell factory for recombinant protein production, AP.TEM

Abstract

Recombinant proteins are highly important for the food, pharmaceutical, and biomedical industries due to their great versatility and applications. Filamentous fungi stand out as excellent producers of these proteins because they can secrete high titers of the protein of interest and perform post-translational modifications that ensure the correct function of the heterologously produced protein. One of the most widely used fungi is Aspergillus oryzae, whose ability to ferment soybeans has been exploited for many years in traditional oriental cuisine. However, one of the bottlenecks in recombinant protein production in filamentous fungi is the simultaneous secretion of various proteases-enzymes capable of cleaving other proteins-which reduces the recombinant protein titer in solution. In this context, the present proposal aims to identify and delete a hypothetical proteolytic core, that is, a set of key proteases secreted by A. oryzae in liquid fermentation. To achieve this, the master's research will rely on secretomic data obtained by various researchers from the FAPESP thematic project 2022/05731-2, titled "BEYOND: Establishing a Fungal Cell Factory for Recombinant Protein Production," in which multiple recombinant proteins (such as ¿-xylosidase, bovine albumin, cutinase, among others) will be produced under standardized conditions-something unprecedented in the literature. The secretomes will be analyzed by mass spectrometry to identify which proteases are produced, and based on this information, the CRISPR-Cas9 system will be used to delete those common to all strains. As proof of concept, the bovine albumin-producing strain will be selected for transformation, allowing a comparison of process yield before and after protease deletion. (AU)