Overcoming prokaryotic toxicity: a SUMO-fused secretory platform in Komagataella phaffii for high-yield production of phage holin Hol41

Phage holin Hol41 is a membrane protein with broad-spectrum antibacterial potential; however, its high hydrophobicity and prokaryotic expression toxicity severely limit its soluble expression and large-scale production. We constructed a synthetic biology-driven secretory expression platform for Hol41 in Komagataella phaffii (K. phaffii) by integrating small ubiquitin-related modifier (SUMO) fusion technology and the α-mating factor secretion pathway. This platform avoids prokaryotic toxicity by exploiting the structural differences between the cell membranes of K. phaffii and Escherichia coli. The SUMO tag combined with α-mating factor-mediated secretion enhances protein solubility, reduces toxicity, and inhibits Hol41 aggregation. After scale-up culture in a 2-L fermenter, the yield of SUMO-Hol41 reached 44.95 mg/L, with a yield coefficient (Yp/s) of 0.00843 mg protein/mg dry cell weight (DCW). MIC and MBC assays revealed that SUMO-Hol41 exhibited selective antibacterial activity against Gram-positive bacteria among seven standard strains: the MIC values against Staphylococcus aureus ATCC 33,951 and Streptococcus pneumoniae ATCC 49,619 were both 128 µg/mL (MBC: 256 µg/mL), and 64 µg/mL (MBC: 128 µg/mL) for Enterococcus faecalis ATCC 29,212. Among Gram-negative bacteria, only Salmonella enteritidis S4 was susceptible (MIC: 128 µg/mL, MBC: 256 µg/mL). Moreover, the purified protein showed dose- and time-dependent antibacterial activity, whose inhibitory effect increased with elevated dosage and prolonged incubation time. This study represents the first successful high-yield functional expression of phage holin Hol41 in K. phaffii. The established K. phaffii-SUMO fusion-secretion strategy effectively overcomes challenges of prokaryotic toxicity and poor solubility, providing a robust platform for developing novel antibacterial agents against drug-resistant bacteria.