Implementation of Metabolic Engineering Tools in Komagataella phaffii for Production of Recombinant Proteins

• Grant number: 25/28489-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2026
• End date: January 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: André Ricardo de Lima Damasio
• Grantee: Sophia Salomão Rozenbaum
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 25/13935-5 - Construction of a Synthetic Expression System in Aspergillus oryzae for Recombinant Protein Production, AP.R

Abstract

The methylotrophic yeast Komagataella phaffii (formerly known as Pichia pastoris) represents one of the most promising biotechnological platforms for the industrial production of recombinant proteins. This unicellular eukaryotic microorganism possesses several exceptional features that make it highly attractive for biotechnological applications, including its ability to grow to high cell densities, utilize methanol as a sole carbon source, perform complex post-translational modifications, and its status as a "Generally Recognized as Safe" (GRAS) organism. In addition, K. phaffii exhibits superior efficiency in the secretion of heterologous proteins compared to other eukaryotic expression systems, making it a valuable alternative to traditional platforms based on Saccharomyces cerevisiae and Escherichia coli.Despite the inherent advantages of K. phaffii as an expression system, several limitations can restrict its maximum production potential. These constraints include bottlenecks in the secretory machinery, limited processing capacity in the endoplasmic reticulum, and limited availability of essential metabolic precursors required for protein biosynthesis. To overcome these barriers and fully exploit the biotechnological potential of this yeast, advanced metabolic engineering strategies are essential for rational optimization of metabolic pathways and cellular processes involved in recombinant protein production.The present project proposes a systematic approach for the development and implementation of metabolic engineering tools in K. phaffii, using the OPENPichia system as a chassis. This system represents a significant advance in the field, offering license-free chassis strains and a modular protein expression toolkit that enables commercial use without intellectual property constraints. The OPENPichia platform is based on the type strain NCYC 2543, which has been modified through truncation of the HOC1 gene, resulting in improved transformability and enhanced secretion efficiency for certain proteins.To validate the effectiveness of the implemented tools, the project will focus on the transformation and expression of two carefully selected target genes. This dual-validation strategy will enable assessment of the system's versatility and efficiency, providing essential comparative data to demonstrate the advantages of the implemented modifications. Ultimately, this project aims not only to establish advanced engineering tools for K. phaffii, but also to complement the research group's existing expertise in Aspergillus spp.-based systems, thereby expanding the range of proteins that can be produced with high efficiency. (AU)