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Background <p>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.</p> Results <p>We constructed a synthetic biology-driven secretory expression platform for Hol41 in <i>Komagataella phaffii</i> (<i>K. phaffii</i>) 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 <i>K. phaffii</i> and <i>Escherichia coli</i>. 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&#xa0;mg/L, with a yield coefficient (<i>Y</i><sub>p/s</sub>) of 0.00843&#xa0;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 <i>Staphylococcus aureus</i> ATCC 33,951 and <i>Streptococcus pneumoniae</i> ATCC 49,619 were both 128&#xa0;µg/mL (MBC: 256&#xa0;µg/mL), and 64&#xa0;µg/mL (MBC: 128&#xa0;µg/mL) for <i>Enterococcus faecalis</i> ATCC 29,212. Among Gram-negative bacteria, only <i>Salmonella enteritidis</i> S4 was susceptible (MIC: 128&#xa0;µg/mL, MBC: 256&#xa0;µg/mL). Moreover, the purified protein showed dose- and time-dependent antibacterial activity, whose inhibitory effect increased with elevated dosage and prolonged incubation time.</p> Conclusions <p>This study represents the first successful high-yield functional expression of phage holin Hol41 in <i>K. phaffii.</i> The established <i>K. phaffii</i>-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.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月27日

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  • Zhichuang Huo,
  • Fangfang Wang,
  • Yanze He,
  • Zhihui Chang,
  • Shasha Feng,
  • Jinzhe Kang,
  • Cuihong Zhong,
  • Yongying Zhang,
  • Yuxiang Shi

摘要

Background

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.

Results

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.

Conclusions

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.

Graphical Abstract