Omics-Based Expression Cassette for Heterologous Protein Production in <i>Y. lipolytica</i>

The nonconventional yeast, <i>Yarrowia lipolytica</i>, is a promising protein expression host, having achieved recombinant protein expression yield on par with the commonly used host, <i>Komagatella phaffii</i> (<i>Pichia pastoris</i>). However, strong, fully constitutive genetic elements and expression cassettes for protein expression in <i>Y. lipolytica</i> remain limited. In this study, we leveraged genome-wide transcriptomics to uncover five strong promoters and four terminators. Among these, the promoter of ribosomal protein L41 demonstrated superior activity to the strongest previously reported promoters. We further demonstrated the functionality of pL41 across different media conditions and by using it to express diverse heterologous proteins. Similarly, we showed that the terminator of glutathione-S-transferase (tGST) supported higher protein expression and low transcriptional readthrough compared to commonly used terminators. To support protein secretion efforts, we utilized a secretomics-guided signal peptide screen to unveil three signal peptides, demonstrating broad applicability to different proteins. Integrating these genetic elements into a new expression cassette (YALI-pSTOmics1) resulted in a 3-fold increase in secretory expression of bovine fibroblast growth factor 2 compared to a combination of the best available state-of-the-art genetic tools for gene expression in <i>Y. lipolytica</i>. This expression cassette represents an open-source alternative to expensive commercial ones. Furthermore, the novel promoters and terminators provide options for metabolic engineering, where reuse of existing genetic parts is often a limitation.