Multi-dimensional engineering enables high-yield secretion of β-galactosidase Bgal1–3 in Komagataella phaffii

Despite its longstanding use in recombinant protein production, the fundamental principles governing signal peptide (SP) functionality in Komagataella phaffii ( K. phaffii ) remain poorly understood. In this study, 64 endogenous SPs were identified from K. phaffii genome and were combined with 52 high-performance exogenous SPs from literature. This SP library was employed to optimize secretion of β-galactosidase Bgal1–3-Y497F, an efficient biocatalyst for producing galactooligosaccharide-enriched and low-lactose dairy products. The characterization identified 35 new SPs, expanding the repertoire of SPs that outperformed the conventional α-mating factor (α-MF) SP by 30 %. The top performers, SP25 and W1, mediated 4.0- and 5.5-fold higher extracellular enzyme activity than α-MF SP, with similar enhancements (2.0–5.5-fold) observed for heterologous proteins β-galactosidase BgaD-D and plastic-degrading enzyme LCC-ICCG. A few key sequence features for efficient secretion were revealed. For N-regions, endogenous SPs preferred +1 charge, whereas exogenous SPs preferred +3 charges. For H-region, both SP types showed bell-curve dependencies (extracellular enzyme activity plotted against hydrophobicity) with a consistent optimal window (hydrophobicity scores = 40–55) that provides a general guideline for SP selection and engineering in this host. Implementing an integrated engineering strategy (W1-based SP engineering, multi-copy strain selection, chaperone co-expression, and cultivation condition optimization), a 17.9-fold production increase was achieved. Scale-up to 5-L bioreactor cultivation yielded 7.0 g/L, the highest reported titer of prokaryotic β-galactosidase in K. phaffii . This work provides both fundamental insights into SP features for efficient protein secretion in K. phaffii and a high-performance industrial strain for dairy processing applications.