Efficient Biosynthesis of Porcine Pancreas Phospholipase A₂ in Engineered <i>Komagataella phaffii</i>

Phospholipase A₂ (PLA₂) is a large enzyme family primarily found in the mammalian pancreas, playing a crucial role in physiological processes by catalyzing the hydrolysis of phospholipids at the <i>sn-2</i> position. Herein, we engineered a <i>Komagataella phaffii</i> X33 as a cell factory for the efficient biosynthesis of porcine pancreas PLA₂ (ppPLA₂). First, we identified the AOX1p promoter and the hybrid signal peptide EXG1-pro as optimal components for ppPLA₂ expression by constructing a secretory signal peptide library (SP-Lib), achieving a ppPLA₂ activity of 510.36 ± 66.57 U/L. Subsequently, a maximum activity of 2.61 × 10³ ± 122.90 U/L was achieved by knockout of the vacuolar sorting receptor VPS10, overexpression of the chaperone protein ERO1, and increased gene dosage of <i>ppPLA</i>₂, approximately 7-fold of the original strain. Finally, in a 3 L bioreactor, ppPLA₂ activity reached 1.83 × 10⁵ ± 469.07 U/L in the engineered strain X33-ppPLA₂-CN2-ΔVPS10-1/2-ERO1, representing a 70-fold increase compared to that in shake flasks. This study provides an efficient strategy for the high-level biosynthesis of ppPLA₂ in <i>K. phaffii</i>, offering valuable insights for expanding ppPLA₂ production to meet the growing demand.