Boosting Recombinant Bovine Chymosin in Komagataella phaffii via Fusion Protein and Constitutive Promoter Expression

Bovine chymosin is key for cheese production, yet its traditional sourcing is unsustainable. While microbial and plant-based alternatives exist, they often cause non-specific proteolysis, leading to bitter flavors in cheese. This study aims to develop a high-yield, methanol-independent platform for recombinant bovine chymosin production by engineering the expression system of <i>Komagataella phaffii</i> through multi-factorial optimization. Initially, the native bovine prochymosin gene (<i>pcw</i>) was codon-optimized (<i>pcm14</i>) and cloned, along with an mCherry-tag construct (<i>clpcm14</i>), into inducible vector pPIC9 for expression in <i>Komagataella phaffii</i> GS115. Screening identified the fusion-tagged strain clp2-91 as the highest producer. Subsequently, the inducible AOX1 promoter in the previously selected clp2-91 strain was replaced with a constitutive GAP promoter, yielding engineered strain GH1. Cultivated in a 3L fermenter, GH1 exhibited a volumetric productivity of 105.03 SU/(mL·h), twice that of inducible strain clp2-91 (53.59 SU/(mL·h)). The further optimization of fermentation conditions (pH 4.0, glucose as carbon source, fed-batch mode) boosted the enzyme activity of GH1 to 12,000 SU/mL. The recombinant chymosin exhibited enzymatic properties similar to those of the native enzyme and, importantly, demonstrated a broader pH stability (pH 2.0-6.0). This study demonstrates an efficient strategy for chymosin expression in <i>K. phaffii</i>, offering insights that may support the future development and optimization of heterologous protein production in this yeast.