Characterizing heterologous protein burden in Komagataella phaffii

Yeast is a widely utilized chassis for heterologous protein production, with Komagataella phaffii well-established as a prominent nonconventional yeast in this field. Despite its widespread recognition, there remains considerable potential to further optimize these cell factories to meet high production demands in a cost-effective and sustainable manner. Understanding the cellular response to the challenges of heterologous protein production can equip genetic engineers with crucial knowledge to develop enhanced strategies for constructing more efficient cell factories. In this study, we explore the molecular response of various K. phaffii strains that produce either the human insulin precursor or Mambalgin-1, examining changes in transcription and changes in intra- and extracellular protein levels. Our findings provide valuable insights into the molecular mechanisms that regulate the behaviour of K. phaffii production strains under the stress of producing different heterologous proteins. We believe that these results will serve as a foundation for identifying new genetic targets to improve strain robustness and productivity. In conclusion, we present new cellular and molecular insights into the response of K. phaffii cell factories to the challenges of burdensome heterologous protein production and our findings point to different engineering strategies for improved cell factory performance.