Multi-level Integrated Engineering Enhances Neutral β-Glucanase Production in Komagataella phaffii

Neutral β-1,3-glucanase (BgA) is a vital enzyme in the food industry, widely used to reduce viscosity in brewing mashes, improve dough rheology in baking, and clarify fruit juices. However, its industrial application is currently hindered by low secretion titers and high production costs. In this study, we established a systematic, multi-layered engineering framework for Streptococcus equinus neutral BgA in the Generally Recognized as Safe (GRAS) host K. phaffii (Komagataella phaffii). Utilizing a modular BioBrick assembly approach, we first performed a combinatorial screening of promoter and signal peptide libraries, identifying P_DAS1 and the alpha-mating factor (MFα) signal peptide as the optimal secretion module, achieving an initial activity of 5.5 U/mL·OD₆₀₀. Subsequently, we implemented a synergistic regulation strategy targeting both transcriptional and translational levels. Co-overexpression of the transcription factors Mxr1 and Mit1 enhanced BgA titers by 72% across strains with varying gene dosages. Further engineering of the translation initiation complex (eIF4G, eIF4E, eIF4A, and Pab1) resulted in a further 1.9-fold increase in yield. To validate the industrial scalability of the engineered strain, high-density fed-batch fermentation was conducted in a 5-L bioreactor, achieving a final BgA titer of 1.1 g/L. This study demonstrates a robust, integrated engineering strategy that effectively resolves secretory bottlenecks, establishing K. phaffii as a competitive platform for neutral β-glucanase production.