BEYOND: establishing a fungal cell factory for recombinant protein production

• Grant number: 22/05731-2
• Support Opportunities: Research Projects - Thematic Grants
• Duration: July 01, 2023 - June 30, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: André Ricardo de Lima Damasio
• Grantee: André Ricardo de Lima Damasio
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Pesquisadores principais: Fábio Márcio Squina ; Rosana Goldbeck Coelho
• Associated researchers: Lucas Miguel de Carvalho ; Marcelo Ventura Rubio ; Sarita Candida Rabelo ; Uffe Hasbro Mortensen
• Associated scholarship(s): 24/03892-4 - Biochemical studies of recombinant PAZymes produced by Aspergillus oryzae engineered strain, BP.DD; 24/02154-0 - Expression of heterologous proteins for cultured meat and plant-based applications, BP.DD; 24/02283-4 - Technical Support for the Development of Plastics Active Enzymes (PAZymes), BP.TT; + associated scholarships 23/12398-0 - PROSPECTION IN PUBLIC DATABASES, CLONING, AND PRODUCTION OF RECOMBINANT HYDROPHOBINS IN A. ORYZAE, BP.IC; 23/08832-7 - Engineering and Characterization of an Aromatic Polyester Degrading Enzyme., BP.IC; 23/12218-2 - Multiplex deletion of secreted proteins in Aspergillus oryzae: a minimum secretome, BP.PD; 22/14112-4 - Development of a collection of extreme thermophilic enzymes active in the conversion of pet plastics, BP.DD; 23/10777-4 - Technical Support for the Development of Plastics Active Enzymes (PAZymes), BP.TT; 21/04254-3 - Development of Biorefinery concepts via coumaric acid conversion into high-value compounds, BP.PD - associated scholarships

Abstract

Filamentous fungi are capable of producing and secreting a variety of enzymes. Aspergillus sp. and Trichoderma sp. are the major fungal cell factories for industrial enzyme production. Aspergillus oryzae is recognized as GRAS (generally regarded as a safe organism) and has the ability to secrete a high titer of proteins. Therefore, A. oryzae has been used as a low-cost microbial cell factory for the production of recombinant proteins and has the potential to be engineered to boost the secretory pathway. The overall goal is to establish the filamentous fungus A. oryzae as a robust and economically viable microbial cell factory for the production of recombinant proteins for different applications. To do that, we want to harness the synthetic biology concept (learn, design, build and test) combining systems biology, fungal genetic engineering, selection of recombinant proteins with biotechnological applications, bioprocess design, and techno-economical analysis. To achieve that, this project is divided into five aims: (Aim 1) Induction of A. oryzae secretory pathway using Brazilian agro-industrial residues; (Aim 2) Removal of host secreted proteins by multiplex gene deletion; (Aim 3) Production of recombinant proteins with biotechnological applications; (Aim 4) Evaluation of recombinant proteins productivity and function; and (Aim 5) Fungal strains fermentation and techno-economic analysis. We propose to work with three groups of proteins: (1) lytic polysaccharide monooxygenases (LPMOs); (2) Plastic-Active enZymes (PAZymes); and (3) alternative proteins applied to the production of plant-based and cultivated meat. Assuming our team integrated work will result in a robust A. oryzae engineered strain producing high titers of different recombinant proteins using an agro-industrial residue as inducer, multiomics experiments (transcriptome, proteome and metabolome) will be carried out to understand how the recombinant strains adapt to high demand for the secretory pathway. These data will be used to build a genome-scale metabolic model (GEM) for A. oryzae, which will allow the planning of new mutations for genetic improvement of the strain. (AU)