Efficient Biosynthesis of Porcine Pancreas Phospholipase A2 in Engineered Komagataella phaffii

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 sn-2 position. Herein, we engineered a Komagataella phaffii 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 ppPLA2, 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 K. phaffii, offering valuable insights for expanding ppPLA2 production to meet the growing demand.