Advances in Key Genetic Elements and Strategies for High-Yield Heterologous Protein Expression in Komagataella phaffii

Komagataella phaffii has emerged as an important eukaryotic microbial cell factory for the production of industrial enzymes, biopharmaceuticals, and food-related proteins owing to its rapid growth, capacity for eukaryotic post-translational modifications, and suitability for high-cell-density fermentation. However, efficient heterologous protein production remains constrained by limitations in transcriptional regulation, protein secretion, and endoplasmic reticulum (ER) protein-folding capacity. This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign (e.g., upstream activating sequence (UAS) duplication and core promoter mutagenesis), signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response (UPR). Rather than focusing on individual expression modules, this review highlights a systems-level engineering perspective that integrates regulatory elements, intracellular processing, and secretory pathway optimization, illustrating the ongoing transition from empirical modification of individual expression elements to systems-level engineering of the secretory pathway. Recent advances in synthetic biology and artificial intelligence (AI)-assisted approaches are further accelerating the development of predictive and precision engineering approaches for K. phaffii. Together, these advances provide a foundation for the rational design of next-generation K. phaffii cell factories for the sustainable production of structurally complex and high-value recombinant proteins.