Cell Wall Engineering of Kluyveromyces lactis for Efficient Extracellular Production of Bovine Chymosin

Abstract Bovine chymosin, an essential milk-clotting enzyme widely utilized in cheese production, was successfully expressed in the generally recognized as safe yeast Kluyveromyces lactis GG799. The recombinant strain SKB was initially constructed. Subsequently, 15 genes involved in the cell wall biosynthesis pathway were knocked out to evaluate their effects on chymosin release. Notably, the double-deletion strain SKB7 (ΔKLLA0_C00803g, ΔKLLA0_F20031g), involving two genes encoding a protein involved in the regulation of cell wall structure/stress response and a cell wall protein, exhibited a 4.5-fold increase in chymosin activity (498 SU/mL) in shake-flask. In fed-batch fermentation, strain SKB7 achieved a high chymosin activity of 4469 SU/mL after 96 h in a 5 L bioreactor. Transcriptomic analysis reveals distinct gene expression in strain SKB7 compared with SKB-3, representing enhanced metabolic robustness and growth potential. This study demonstrates that cell wall engineering is an effective strategy for improving chymosin production in K. lactis.